Development of gut microbiota in the pig: modulation

UNIVERSITAT AUTONÒMA DE BARCELONA
Development of gut microbiota in the pig: modulation of
bacterial communities by different feeding strategies.
MEMÒRIA PRESENTADA PER MARIA SOLEDAD CASTILLO GÓMEZ
PER ACCEDIR AL GRAU DE DOCTOR DINS EL PROGRAMA DE DOCTORAT DE
PRODUCCIÓ ANIMAL DEL
DEPARTAMENT DE CIÈNCIA ANIMAL I DELS ALIMENTS
BELLATERRA, MARÇ DE 2006
Susana M. Martín-Orúe, investigadora del departament de Ciència Animal i dels
Aliments de la Facultat de Veterinària de la Universitat Autònoma de Barcelona,
certifica:
Que la memòria titulada “Development of gut microbiota in the pig: modulation of
bacterial communities by different feeding strategies”, presentada per Maria Soledad
Castillo Gómez per optar al grau de Doctor en Veterinària, ha estat realitzada sota la
seva direcció i, considerant-la acabada, autoritza la seva presentació per que sigui
jutjada per la comissió corresponent.
I per que consti als efectes oportuns, signa la present a Bellaterra, 20 de Març de 2006.
Dra. Susana M. Martín-Orúe.
The author was in receipt of a grant from the Departament d’Universitats, Recerca i
Societat de la informació (DURSI) of the Generalitat de Catalunya for this study.
Sin duda, en la elaboración de una tesis y en el periodo de formación predoctoral
participan muchas personas además del doctorando, por ello, quiero en primer lugar,
agradecer el apoyo recibido durante estos años.
En primer lugar, al equipo docente e investigador del grup de Nutrició, en especial a
Susana, gracias por tu apoyo y ayuda durante estos años. Josep, Francisco, Mariola,
AnaCris y Roser, gracias por brindarme la oportunidad de formar parte del equipo de
Nutrición, por vuestra colaboración, consejos y cafés que hemos compartido. Y, sobre
todo a Olga, muchísimas gracias por tu apoyo.
A todos los becarios, aquellos que me brindaron su ayuda y consejos cuando
empezaba (Joaquin, Jaume, Dani, Lucía), y a todos los demás con los que he
compartido estos años: Montse, Eva, Alba, Núria, Edgar, Ceci, Arantza, Marta, Gabri,
Walkiria, Juan Carlos, José, Sandra, Carol, Muzzafer, Francesc, gràcies!!!, A todos con
los que he compartido despacho, Aina, Luciano, Glaubert, Feliu, Vincent…També als
altres becaris amb els que he compartit l’aventura del doctorat: Mercè, Miquel, Maribel,
Montse, Anna, Laura, gràcies!
Also, I would like to thank to the Gut microbiology and Immunology division of the
Rowett Research Institute, in particular to Harry Flint, Sylvia Duncan and Gail Skene,
for their collaboration and support; as well to the rest of the staff and students who
shared with me the adventure of the FISH, thanks a lot!!
With special regards to Justin and Vanessa (and the little Maria Isabella), because
they were my american family in Aberdeen, Scotland.
Guillem, Pablo, David, Àlex, Joan, Beti, Anna i Laura, per ser els de sempre; Javier,
por estar siempre, gracias.
Muy especialmente a mi familia por su ayuda y apoyo incondicional, sin vosotros
esta tesis no hubiera sido posible. Y, por supuesto a todos aquellos que en algún
momento han sido cómplices en la elaboración de este trabajo, gracias.
PARA MIS PADRES,
Resumen
RESUMEN
El objetivo de esta tesis fue el estudio de la microbiota gastrointestinal porcina, para
mejorar el conocimiento existente de este complejo ecosistema y así, ayudar de alguna
forma en el desarrollo de nuevas estrategias alimentarias para sustituir los antibióticos
promotores del crecimiento recientemente prohibidos en la Unión Europea.
Para alcanzar este objetivo, se diseñaron diferentes pruebas experimentales (capítulo
4-9). En la Prueba I, se desarrolló la técnica de PCR cuantitativa para cuantificar
bacterias totales, lactobacilli y enterobacteria en muestras de contenido digestivo. Con
el fin de validar su utilidad, los resultados obtenidos se compararon con los que se
obtuvieron con métodos tradicionales (cultivo en medio selectivo para lactobacilli y
enterobacteria, y microscopía directa para bacterias totales). La PCR mostró valores
superiores, en términos de copias del gen 16S rRNA que la microscopía directa y los
cultivos. Sin embargo, a pesar de la diferencia, la ratio lactobacilli:enterobacteria fue
similar entre métodos. Diferentes motivos pueden estar detrás de la diferencia entre
métodos, tanto una sobreestimación con la PCR como una subestimación con los
métodos tradicionales. No obstante, los contajes para el total de bacterias y lactobacilli
mostraron una correlación significativa. Por ello, este método se consideró como válido
para cuantificar cambios bacterianos en el tracto gastrointestinal del cerdo.
Con el fin de estudiar el establecimiento de la microbiota en el cerdo tras el destete,
se diseñó la Prueba II. En ésta, 12 lechones (20 ± 2 días) de 6 camadas diferentes
fueron divididos en un grupo control, el cual permaneció con la madre, y un grupo
experimental el cual fue destetado y alimentado con una dieta pre-starter commercial.
Tras una semana, los animales fueron sacrificados y se recogieron muestras de
contenido de ciego. Para estudiar el cambio en la microbiota, se cuantificó el total de
bacterias, lactobacilli y enterobacterias mediante PCR a tiempo real. Además, para
obtener una imagen global del cambio producido por el destete, se utilizó la técnica del
t-RFLP (“Terminal restriction fragment length polymorphism”). La población total
bacteriana, así como la biodiversidad, medida como número de bandas obtenidas por tRFLP, fue similar entre grupos, pero hubo un descenso importante en el ratio
lactobacilli:enterobacteria. Además, el análisis de similaridad de los perfiles obtenidos
i
Resumen
por t-RFLP, mostró una agrupación separada de los grupos experimentales. Inferiendo
con los fragmentos teóricos se observaron diferencias entre grupos. Los cerdos lactantes
mostraron una mayor diversidad de fragmentos compatibles con bacterias ácido lácticas
y se observó la presencia de algunos picos compatibles con Clostridium coccoides, C.
butyricum y Lactobacillus delbruekii que no se encontraron en los animales destetados.
Estos resultados confirman el destete como un punto crítico en el establecimiento de la
microbiota gastrointestinal.
En la Prueba III, se utilizaron cerdos en crecimiento para estudiar la microbiota
gastrointestinal y a su vez, el potencial de la fibra para modificar este ecosistema. Para
ello, 32 cerdos (15 ± 0.38 kg de peso vivo) se distribuyeron en 4 tratamientos: una dieta
control, una dieta rica en almidón resistente por la inclusion de maíz con un mayor
tamaño de particula, una dieta rica en polisacáridos no amiláceos solubles por la
inclusion de pulpa de remolacha, y una cuarta dieta rica en polisacáridos no amiláceos
insolubles por la inclusion de salvado de trigo. Tras seis semanas de alimentación ad
líbitum, los animales fueron sacrificados y el contenido digestivo fue muestreado. La
técnica del FISH (“Fluorescent in situ hybridization”) se utilizó con el fin de describir
los grupos bacterianos mayoritarios a lo largo del tracto gastrointestinal. Se utilizaron
diferentes sondas para cuantificar bacterias pertenecientes al Bacteroides/Prevotella
grupo, Ruminococcus flavefaciens, Ruminococcus bromii, clostridia cluster IV,
clostridia cluster IX, Streptococcus/Lactococcus y Lactobacillus/Enterococcus sp. en
estómago, yeyuno distal, colon proximal y recto. Los resultados obtenidos revelaron
marcadas diferencias en la composición de estos grupos a lo largo del tracto, que no
fueron marcadamente afectados por la dieta. En estómago, streptococci y lactobacilli
fueron los grupos predominanates, mientras que en intestino grueso,
el grupo de
Bacteroides/Prevotella, clostridial cluster XIVa, IV, y ruminococci fueron los más
abundantes. Los resultados obtenidos por RFLP (“restriction fragment length
polymorphism”) mostraron cambios en el perfil bacteriano dependiendo de la dieta
administrada. Los animales que recibieron salvado de trigo mostraron una menor
biodiversidad con unos perfiles más similares entre animales. Además, se hallaron
cambios en la fermentación mediante la determinación de ácidos grasos volatiles; Las
dietas ricas en polisacáridos no amiláceos mostraron una menor concentración de ácidos
grasos ramificados y valerico.
ii
Resumen
En las pruebas IV y V, se estudiaron diferentes aditivos comerciales como posibles
alternativas a los antibióticos promotores del crecimiento, con especial interés en sus
efectos en la microbiota gastrointestinal. En concreto, en la Prueba IV, se testaron 3
aditivos: avilamicina (como control positivo), butirato sódico y un extracto de plantas
(carvacrol, cinamaldehido y capsicum). Un total de 40 (18-22 días) cerdos se
distribuyeron en cuatro tratamientos: una dieta control, ésta con 0.04% de avilamicina,
con 0.3% de butirato sódico o con 0.03% de extracto de plantas. Después de dos
semanas los animales fueron sacrificados y el contenido digestivo fue muestreado.
Como en las pruebas anteriores, la PCR a tiempo real se utilizó para estudiar los
cambios en la microbiota. No se encontraron diferencias en el total de bacterias a lo
largo del tracto gastrointestinal con ninguna de las dietas, aunque la ratio
lactobacilli:enterobacteria en ciego fue superior para los animales que recibieron el
extracto de plantas. La técnica del RFLP mostró diferencias en el perfil bacteriano,
agrupando los animales en función de la dieta administrada. La actividad bacteriana
total medida como bases púricas también mostró diferencias entre dietas. Estos
resultados podrían indicar que el efecto de los diferentes aditivos testados no se debería
a una reducción en el total de bacterias sinó a modificaciones en la composición y
actividad de la microbiota.
Finalmente, en la Prueba V, una fuente comercial de mananoligosacáridos y de zinc
orgánico, administrados por separado o conjuntamente fueron testados para mejorar los
indices productivos, microbiota gastrointestinal y respuesta inmune. En este caso, 128
cerdos (18-22 días de vida) se distribuyeron en cuatro tratamientos: una dieta control,
ésta dieta con 0.2% de mananoligosacáridos, con 0.08% de zinc orgánico o con ambos
aditivos. Las dietas fueron administradas durante cinco semanas. Tras dos semanas, 32
animales fueron sacrificados y el contenido digestivo fue muestreado. Se observó una
mejora en el índice de conversion para todo el periodo experimental cuando los dos
aditivos se añadieron conjuntamente. Los mananoligosacáridos redujeron la
enterobacterias en yeyuno. La adición de zinc orgánico, tendió a incrementar el peso en
vacío del ileon, que fue considerado como el segmento de intestino delgado con placa
de Peyer continua. Estos resultados sugieren diferentes mecanismos de acción de los
aditivos, mientras que los mananoligosacáridos podrían estar actuando modulando el
ecosistema bacteriano, mediante la inhibición de algunos grupos, el incremento en el
peso vacío del ileon podría sugerir un efecto inmunológico. Además, el efecto positivo
iii
Resumen
en la ratio vellosidad:cripta cuando ambos aditivos se incluyeron conjuntamente podría
indicar acciones complementarias.
Los resultados obtenidos en la presente tesis demuestran la validez de diferentes
métodos moleculares para el studio de la microbiota gastrointestinal del cerdo.
Ecosistema muy instable durante las primeras edades, con un cambio drástico al destete
pero que consigue una estabilización de los grupos mayoritarios en el animal adulto. Por
otra parte, los efectos promotores de las diferentes alternativas testadas parecen estar
relacionados con sutiles cambios en la microbiota gastrointestinal más que con drásticos
efectos antimicrobianos. No obstante, en algunos casos (butirato sódico, zinc) otros
efectos diferentes al microbiano podrían estar implicados.
iv
Summary
SUMMARY
The main objective of this thesis was to study pig gut bacteria to improve our
knowledge of this complex ecosystem as this could help in the development of new feed
strategies to substitute antibiotics as growth promoters.
To achieve this main objective, a set of five trials were designed (chapter 4-8). In
Trial I, real-time PCR was developed to quantify total bacteria, lactobacilli and
enterobacteria in digesta samples. To validate its usefulness, results obtained were
compared to those obtained by traditional methods (selective culture for lactobacilli and
enterobacteria, and direct microscopy for total bacteria). Real time PCR showed higher
values in terms of 16S rRNA gene copies than direct microscopy counts or CFU.
Despite the differences, the lactobacilli:enterobacteria ratio was similar between
methods. Differences between methods might caused by an overestimation with PCR by
quantification of dead bacteria or free DNA, and also an underestimation with
conventional methods. Values obtained by PCR and traditional methods showed a
significant correlation for lactobacilli and total bacteria. Therefore, real-time PCR was
considered a valid method to quantify microbial shifts in the gastrointestinal tract.
To study pig gut microbiota establishment in the young pig after weaning, trial II
was designed. Twelve pigs (20 ± 2 days ) from 6 different litters were divided into a
control group that remained with the sow and an experimental group that was weaned
and fed a commercial post-weaning diet. After one week, the animals were sacrificed
and samples from cecal digesta were taken. To assess microbial shift, total bacteria,
lactobacilli and enterobacteria were quantified using real-time PCR. To achieve an
overall picture of the change in the global microbial profile, terminal restriction
fragment length polymorphism of the PCR amplified 16S rRNA gene was applied.
Total bacteria and biodiversity of the microbial ecosystem were similar between both
experimental groups, although there was a decrease in the lactobacilli:enterobacteria
ratio. Also, cluster analysis grouped animals in two different clusters. Considering
theoretical restriction fragment lengths, differences in compatible bacterial groups were
observed between groups. Suckling pigs showed a higher lactic acid bacteria diversity.
Also peaks compatible with Lactobacillus delbruekii, Clostridium coccoides and C.
v
Summary
butyricum were also mostly present in suckling pigs. Results therefore confirm weaning
as a challenging point on the indigenous microbiota establishment.
In Trial III, growing pigs were used to study pig gut microbiota and the potential of
fiber to modify this ecosystem. A total of 32 pigs (15 ± 0.38 kg of body weight) were
distributed into four experimental diets: a control diet, a diet enriched in resistant starch
by inclusion of coarse-ground corn, a diet enriched in soluble fiber by addition of 8 %
sugar beet pulp and a diet rich in insoluble fiber by inclusion of 10 % wheat bran. After
six weeks of feeding ad libitum, animals were sacrificed and samples of digesta content
were taken. Fluorescent in situ hybridization (FISH) was applied to describe main
bacterial groups along the gastrointestinal tract and to detect changes related to the diets.
Probes to detect changes in total bacteria, Bacteroides/Prevotella group, Ruminococcus
flavefaciens, Ruminococcus bromii, clostridia cluster IV, clostridia cluster IX,
Streptococcus/Lactococcus and Lactobacillus/Enterococcus sp. were used in samples
from the stomach, distal jejunum, proximal colon and rectum. FISH revealed marked
differences in the composition of the microbiota throughout the gastrointestinal tract,
which were relatively unaffected by changes in the diet. Streptococci and lactobacilli
were predominant in the stomach whereas Bacteroides/Prevotella, clostridial cluster
XIVa, IV, and ruminococci were predominant in the lower tract. Restriction fragment
length polymorphism (RFLP) profiles showed changes in the bacterial profile related to
diet, with pigs fed a wheat bran showing the lowest biodiversity and also having the
most similar patterns. Moreover, changes in fermentation activity were detected when
short-chain fatty acids were measured. Diets rich in non-starch polysaccharides (wheat
bran and sugar beet pulp) showed lower molar percentages of branched chain fatty acids
and valeric acid.
In trials IV and V, different commercial additives were studied as potential
alternatives to antibiotic growth promoters, paying special attention to their effects on
gut microbiota. In particular, in Trial IV, three different additives were tested:
avilamycin (as a positive control), sodium butyrate and a commercial plant extract
(carvarol, cinnamaldehyde and capsicum). Forty early-weaned (18 to 22 d) pigs were
distributed into four dietary treatments: a control diet, a diet with 0.04% avilamycin, a
diet with 0.3% sodium butyrate or with 0.03% plant extract mixture. After two weeks,
the animals were sacrificed and samples from digesta were taken. As in the previous
trials, real-time PCR was used to assess microbial shifts. The total microbial load did
not show differences between diets, although, there was an increase in the
vi
Summary
lactobacilli:enterobacteria ratio in the cecum of piglets fed with plant extracts. RFLP
also showed differences in microbial profile in jejunum digesta samples, with an
increase in biodiversity with the different additives compared to control diet. Total
microbial activity measured as purine bases also showed differences between diets. In
the light of these results, the effect of the different additives would not be related to a
reduction in the toal bacterial load, but rather to changes in the ecological structure and
metabolic activity of the microbial community.
Finally, in Trial V, a commercial source of mannan-oligosaccharides and organic
zinc, offered alone or in combination, were evaluated to enhance performance,
gastrointestinal health and immune response. A total of 128 early-weaned pigs (18 to 22
d) were distributed into four dietary groups. For five weeks, animals received either a
control diet, a diet with 0.2 % mannan-oligosaccharides, a diet with 0.08 % zinc-chelate
or a diet with both additives together. Two weeks after weaning, 32 animals were
sacrificed and digesta samples were taken to study the effect of the additives on gut
health and immunity. An improvement in feed:efficiency was observed with both
additives for the whole period. Mannan-oligosaccharides reduced enterobacteria counts
in jejunum. The addition of organic zinc tended to increase empty ileal weight, defined
as the segment including the continuous Peyer’s patch, and crypt depths were lower in
the animals offered both additives together. These results suggest different modes of
action of the additives tested; whilst mannan-oligosaccharides might be acting by
modulation of intestinal microbiota through inhibition of certain microbial groups, the
observed increase in ileal weight with zinc suggests a possible immunological effect. In
addition, the response observed in gut architecture may be behind complementary
actions when both additives were added together.
Results obtained show the usefulness of different molecular methods for studying
pig gut microbiota quantitatively and qualitatively. This ecosystem, as confirmed, is
specially unstable during the first weeks of life with marked changes at weaning.
However, colonization progresses, resulting in a relatively stable composition in the
main bacterial groups in the adult pig. The effect of the different additives tested might
be related to subtle changes in microbiota composition more than drastic antimicrobial
effects. Moreover, other effects not directly related with microbiota might be involved.
vii
Abbreviations used
ABBREVIATIONS USED
DM: dry matter
DNA: deoxy nucleic acid
dNTP: deoxy-nucleotide-triphosphate
E:L: ratio enterobacteria:lactobacilli
ENT: enterobacteria
F-ent: forward primer for enterobacteria
FISH: fluorescent in situ hybridization
F-lac: forward primer for lactobacilli
FM: fresh matter
F-tot: forward primer for total bacteria
G: guanine
G:F: gain feed ratio
GALT: gut associated lymphoid tissue
GC: diet enriched in resistant starch
(Trial III)
GIT: gastrointestinal tract
HPLC: high performance liquid
cromatography
IEL: intraepithelia limphocyte
iNSP: insoluble non-starch
polysaccharides
L:E: ratio lactobacilli:enterobacteria
LACT: lactobacilli
MAC: microflora associated
characteristic
NOD: nucleotide-binding
oligomerization domain
NSP: non-starch polysaccharides
OM: organic matter
P: P-value
PAMP: pathogen-associated molecular
pattern
PAS: periodic acid Schiff reaction
PB: purine bases concentration
A: adenine
AB: diet containing 0.04% avilamycin
(Trial IV)
AC: diet containing 0.3% sodium
butyrate (Trial IV)
ADFI: average daily feed intake
ADG: average daily gain
AGP: antibiotic growth promoter
BCFA: branched chain fatty acids
BD: below detection
BM: diet containing 0.2% mannanoligosaccharides (Trial V)
BMP: diet containing 0.2% mannanoligosaccharides plus 0.08% organic
zinc (Trial V)
bp: base pair
BP: diet enriched in soluble fiber (Trial
III)
BP’: diet containing 0.08% organic zinc
(Trial V)
BSA: bovine serum albumin
BW: body weight
C: cytosine
CD: crypt depth
CECT: colección española de cultivos
tipo
CFB: cytophaga-flexibacter-bacteroides
phylum
CFU: colony forming unit
CP: crude protein
CT: control diet
DAPI: 4’,6’-diamino-2-phenylindole
DGGE: denaturant gradient gel
electrophoresis
ix
Abbreviations used
PBS: posphate buffered saline
PCR: polymerase chain reaction
PRR: pattern recognition receptor
qPCR: quantitative polymerase chain
reaction
R-ent: reverse primer for enterobacteria
RFLP: restriction fragment length
polymorphism
R-lac: reverse primer for lactobacilli
RNA: riboncleic acid
RS: resistant starch
R-tot: reverse primer for total bacteria
S: suckling pigs
SCFA: short chain fatty acids
SD: standard desviation
SEM: standard error of the mean
sNSP: soluble non-starch
polysaccharides
T: tymine
TGGE: temperature gradient gel
electrophoresis
TLR: toll-like receptor
TRF: terminal restriction fragment
t-RFLP: terminal restriction fragment
length polymorphism
W: weaned pigs
WB: diet enriched in insoluble fiber
(Trial III)
XT: diet containing 0.03% plant extract
mixture (Trial IV)
16S rRNA: ribosomal small sub-unit
x
Index
INDEX
Chapter 1. General introduction
p. 1
Chapter 2. Literature review
2.1. Development of the intestinal microbiota after birth
2.1.1.First colonizers
2.1.2.Weaning: the adaptation to dry food
2.1.3.Autochtonous microbiota in the adult pig
2.2. Main functions of the indigenous microbiota in the gut
2.2.1.Effects of indigenous bacteria on gut maturation and
development
2.2.2.Establishment of the gut barrier and colonization resistance
2.2.2.1.Glycoconjugates of the mucosa as specific
attachment site
2.2.2.2.Molecules involved in bacterial adhesion
2.2.3.Effects of indigenous microbiota on immune response
2.2.3.1.Commensal bacteria tolerance-ignorance
2.2.4.The role of microbiota on digestion and absorption of nutrients
2.2.4.1.Carbohydrate utilization by indigenous bacteria
2.2.4.2.Protein utilization by indigenous bacteria
2.2.4.3.Lipid utilization by indigenous bacteria
2.3. Modulation of intestinal equilibrium through the feed
2.3.1.Macro-indredients
2.3.1.1.The role of dietary fiber
2.3.1.2.Fermented liquid feed
2.3.2.Micro-ingredients and in-feed additives
2.3.2.1.Prebiotics
2.3.2.2.Probiotics
2.3.2.3.Symbiotics
2.3.2.4.Acidifiers
2.3.2.5.Minerals: zinc and copper
2.3.2.6.Plant extracts
2.3.2.7.Other additives
2.4. New tools for the analysis of the gastrointestinal microbiota
2.4.1.Quantitative techniques
p. 4
p. 6
p. 6
p. 9
p. 12
p. 16
xi
p. 16
p. 18
p. 20
p. 22
p. 23
p. 24
p. 26
p. 28
p. 29
p. 30
p. 32
p. 32
p. 33
p. 37
p. 39
p. 39
p. 40
p. 42
p. 42
p. 44
p. 45
p. 46
p. 48
p. 49
Index
2.4.1.1.Quantitative Polymerase Chain Reaction
2.4.1.2.Fluorescent In Situ Hybridization
2.4.2.Fingerprinting techniques
2.4.2.1.Denaturant/Temperature Gradient Gel Electrophoresis
2.4.2.2.Terminal Restriction Fragment Length Polymorfism
p. 49
p. 52
p. 56
p. 56
p. 59
Chapter 3. Objectives
p. 62
Chapter 4. Trial I. Quantification of total bacteria, enterobacteria and
lactobacilli populations in pig digesta by real-time PCR
4.1. Introduction
4.2. Material and methods
4.2.1.Sample preparation
4.2.2.Bacteria quantification by traditional methods
4.2.3.Bacteria quantification by real-time PCR
4.3. Results and discussion
4.4. Conclusion
p. 66
p. 68
p. 68
p. 68
p. 69
p. 69
p. 71
p. 75
Chapter 5. Trial II. Influence of weaning on caecal microbiota of pigs: use of
real-time PCR and t-RFLP
5.1. Introduction
5.2. Material and methods
5.2.1.Animals and housing
5.2.2.Sacrifice and sampling
5.2.3.Statistical analysis
5.3. Results and discussion
5.3.1.Bacterial quantitative change measured by real-time PCR
5.3.2.Ecological bacterial changes, t-RFLP results
5.4. Conclusions
p. 76
p. 78
p. 79
p. 79
p. 79
p. 83
p. 83
p. 83
p. 85
p. 91
Chapter 6. Trial III. Molecular analysis of bacterial communities along the
pig gastrointestinal tract
6.1. Introduction
6.2. Material and methods
6.2.1.Animals and diets
6.2.2.Sample collection and processing
6.2.3.Statistical analysis
p. 92
p. 94
p. 95
p. 95
p. 95
p. 99
xii
Index
6.3. Results
6.3.1.Microflora structure along the gastrointestinal tract as
analyzed by FISH
6.3.2.Effects of fibre on microbial composition as estimated by
RFLP and fermentation profiles
6.4. Discussion
6.5. Conclusions
p. 99
p. 99
p. 100
p. 103
p. 106
Chapter 7. Trial IV. The response of gastrointestinal microbiota to the use of
avilamycin, butyrate and plant extracts in early-weaned pigs
7.1. Introduction
7.2. Material and methods
7.2.1.Animals and housing
7.2.2.Dietary treatments and feeding regime
7.2.3.Collection procedures and measurements
7.2.4.Statistical analysis
7.3. Results
7.3.1.Chages in the total microbial counts
7.3.2.Changes in the microbial ecosystem
7.3.3.Changes in metabolic bacterial activity
7.4. Discussion
7.5. Implications
p. 108
p. 110
p. 110
p. 111
p. 111
p. 113
p. 116
p. 118
p. 118
p. 120
p. 122
p. 124
p. 128
Chapter 8. Trial V. Use of mannan-oligosaccharides and zinc chelate as growth
promoters and weaning preventative in weaning pigs: effects on
microbiota and gut function
8.1. Introduction
8.2. Material and methods
8.2.1.Animals and diets
8.2.2.Performance and collection procedures
8.2.3.Analytical methods
8.2.4.Statistical analysis
8.3. Results
8.3.1.Growth performance
8.3.2.Faecal consistency
8.3.3.Organ weights and small intestine length
8.3.4.Short chain fatty acids
p. 129
p. 131
p. 132
p. 132
p. 132
p. 133
p. 134
p. 136
p. 136
p. 137
p. 138
p. 138
xiii
Index
8.3.5.Quantitative changes in microbial population
8.3.6.Immune proteins and intestinal morphology
8.4. Discussion
8.4.1.Changes on microbial ecosystem
8.4.2.Effect on gut function
8.5. Implications
p. 140
p. 141
p. 141
p. 142
p. 145
p. 147
Chapter 9. General discussion
9.1. Usefulness of quantitative PCR, FISH and t-RFLP to study the
intestinal microbiota
9.2. Weaning: a critical stage in the indigenous pig microbiota
establishment
9.2.1.Establishment of adult gut bacteria
9.3. Are antibiotics-growth promoters a model to copy?
9.3.1.Mode of action of antibiotics: quantitative or qualitative
effects on gut microbiota?
9.3.2.Other in-feed additives with antimicrobial properties
9.3.3.Effects on microbiota by other mechanisms
9.3.4.Other strategies to improve health and promote growth
9.4. Summary
p. 162
p. 163
p. 165
p. 167
p. 168
Chapter 10. Conclusions
p. 171
Chapter 11. Literature cited
p. 174
xiv
p. 150
p. 152
p. 158
p. 161
p. 162
Index
Figures index
Chapter 2
Fig. 2.1. (A) Evolution of aerobic and anaerobic bacteria in piglet feces
p. 8
from birth to 120 days of life
Fig. 2.1. (B) Total bacteria counts and percentage of coliforms,
Bacteroides and Clostridium in piglet feces (Swords et al., 1993)
Fig. 2.2. Review diagram of piglets post-weaning challenge
p. 8
p. 10
Fig. 2.3. (A) Diversity of the intestinal microbiota in piglets from birth
to 14 days afer weaning.
p. 11
(B) Dendogram based on TGGE profiles of one piglet (Inoue et al.,
2005)
p. 11
Fig. 2.4. Intestinal immune geography of responses to commensal bacteria
(Macpherson et al., 2005)
p. 27
Fig. 2.5. Representation of real-time PCR with TaqMan (A) and SYBR
Green (B)
p. 51
Fig. 2.6. (A) Epifluorescent image of mixed culture of seven
Bifidobacterium species by multi color FISH
p. 55
(B) Identification of Bifidobacterium species in human fecal
samples using multi color FISH (Takada et al., 2004)
p. 55
Fig. 2.7. PCR-DGGE profile generated from fecal samples obtained
from a piglet over a 20-day experimental period using primers
specific for the V3-16S rDNA (Simpson et al., 2000)
p. 58
Fig. 2.8. An example of fragments and visualization of the
electropherogram obtained after an enzymatic restriction
p. 60
Fig. 2.9. T- RFLP profiles obtained from cecum digesta in pigs receiving
different doses of zinc oxide and copper sulphate (Höjberg et al.,
2005)
p. 61
Chapter 4
Fig. 4.1. Bacterial loads in jejunum dgesta of pigs as total bacteria,
lactobacilli or enterobacteria measured by qPCR, DAPI
staining or selective culture technique
xv
p. 73
Index
Fig. 4.2. Correlation between the number of total bacteria measured by
qPCR or by DAPI in jejunum digesta samples
p. 74
Chapter 5
Fig. 5.1. Bacterial loads in the caecum of suckling and weaned pigs
measured by quantitative PCR
p. 84
Fig. 5.2. Dendogram illustrating the effect of weaning in t-RFLP
banding patterns
p. 87
Fig. 5.3. Electropherogram produced from Hha I digestion of 16S
rRNA PCR products from one suckling and one weaned piglet
p. 87
Chapter 6
Fig. 6.1. Ecological changes in microbial population measured by RFLP.
Dendogram illustrating the percentage of similarity of PCR
-RFLP banding patterns in samples of proximal colon digesta
p. 101
Fig. 6.2. Fermentation patterns (pH, SCFA and purine bases concentration)
in the proximal colon digesta from experimetal pigs
p. 101
Chapter 7
Fig. 7.1. Quantitative PCR for total bacteria. (A) The amplification plot of
the standards used to quantify total bacteria
p. 118
(B) DNA concentrations plotted vs. Thereshold cycle value to
construct the standard calibration curve
p. 119
(C) Bacterial loads in the stomach, jejunum, cecum, distal colon
colon digesta and in the jejunum mucous layer measured by
quantitative PCR in early-weaned pigs
p. 119
Fig. 7.2. Ecological changes in microbial population of jejunum digesta
measured by RFLP. (A) Example of gel electrophoresis of
the PCR amplifiedV3, V4 and V5 regions of the 16S rDNA
restricted with the enzyme Hha I
p. 121
(B)Dendogram illustrating the correlation between experimental
diets in PCR-RFLP banding patterns
Fig. 7.3. Purine bases concentration in samples from the ileum, cecum,
xvi
p. 122
Index
proximal colon, distal colon and rectum in weaned pigs receiving
A control diet (CT) or the same diet with avilamycin (AB), butyric
acid (AC) or a plant extract mixture (XT)
p. 123
Chapter 8.
Fig. 8.1. Voluntary feed intake of pigs receiving a control diet (CT),
the same diet supplemented with Bio-Mos (BM), Bioplex-Zn
(BP) or both additives (BMP) the first week post-weaning
p. 136
Fig. 8.2. Faecal consistency in pigs receiving a control diet (CT),
or the same diet supplemented with Bio-Mos (BM) Bioplex-Zn
(BP) or both additives during three weeks after weaning
p. 138
Chapter 9
Fig. 9.1. Melting curve oftained after the PCR reaction for total (A),
enterobacteria (B) and lactobacilli (C)
Fig. 9.2. Pie chart with the major 5'-terminal fragments expressed as
the mean of the percentage of the toal area in suckling (S)
and weaned (W)group
Fig. 9.3. Total bacteria, Bacteroides/Prevotella group, clostridia cluster
XIVa, F. prausnitzii, R. flavefaciens and R. bromii,
clostridia cluster IX, Streptococcus/Lactococcus spp. and
Lactobacillus/Enterococcus spp. measured by FISH
xvii
p. 154
p. 160
p. 162
Index
Tables index
Chapter 2
Table 2.1. Main bacteria traditionally cultured from the pig gut
(adapted from Stewart et al., 1999)
Table 2.2. Major phylogenetic lineages to which the phylotypes from the pig
gastrointestinal tract were affiliated (Leser et al., 2002)
Table 2.3. Number of cellulolytic bacteria from fecal samples of sows fed
diets containing various levels of fiber (Varel and Pond,, 1985)
Table 2.4. Selected bacterial counts (CFU/g wet weight) in the colon
of weaned pigs fed diets containing different sources of starch
(MacFarland, 1998, reviewed by Hillman, 2001)
Table 2.5. Some probes used actually to quantify different gastrointestinal
Bacteria
p. 13
p. 14
p. 35
p. 36
p. 54
Chapter 5
Table 5.1. Control diet composition (as fed basis) administered to pigs
p. 79
Table 5.2. Theroretical restriction 5' fragment length predicted for the major
pig gut bacteria.
Chapter 6
Table 6.1. Sequences of oligonucleotides used in the study
Table 6.2. (A) Fermentation parameters including pH, and purine bases
Concentration in samples of proximal colon contents from
pigs receiving experimental diets
(B) SCFA profile in digesta of proximal colon
Table 6.3. Proportions of specific bacterial groupings in different regions
of the porcine digestive tract estimated by FISH
Chapter 7
Table 7.1. Control diet composition, as fed basis
Table 7.2. Material and conditions for the quantification of total bacteria,
enterobacteria and lactobacilli in digesta simples
Bacterial populations, size of lactobacilli, and enterobacteria in
Table 7.3. the
distal jejunum and cecum measured by qPCR in early-weaned
pigs
Table 7.4. Bacterial enzymatic activity in samples of the cecum and colon
xix
p. 82
p. 98
p. 101
p. 101
p. 112
p. 116
p. 120
Index
contents from early-weaned pigs
Chapter 8
Table 8.1. Composition as fed basis of pre-starter and starter control
diets of phase 1 and phase 2
Initial and final pig body weight (kg), voluntary feed intake
Table 8.2. (kg/day)
average daily gain (kg/day) and feed efficiency in weaned pigs
Table 8.3. Weight (g/kg BW) and length (m) of different parts of the gut
from early-weaned pigs sacrificed two weeks post-weaning
Table 8.4. pH, SCFA and lactic acid contentration in the stomach, ileum,
and caecum of pigs sacrificed two weeks post-weaning
Table 8.5. Purine bases concentration in the ileum, caecum and rectum, and
bacterial populations (lactobacilli and enterobacteria) from the
jejunum measured by real-time PCR in ileum digesta in pigs
Table 8.6. Plasma and ileal immunoglobulin (IgA, IgM, IgG) concentration
and jejunum histological parameters in weaned pigs sacrificed
two weeks post-weaning
Chapter 9
Table 9.1 Results of lactobacilli:enterobacteria ratio and its relation with
performance from pigs included in trial II, IV and V
Table 9.2. Summary of the main effects found in the different additives
tested in the trials included in the thesis
p. 124
p. 135
p. 137
p. 139
p. 140
p. 143
p. 144
p. 155
p. 169
TRIAL IV
TRIAL III
TRIAL II
TRIAL I
1
OBJECTIVES
GENERAL INTRODUCTION
TRIAL V
LITERATURE
REVIEW
INTRODUCTION
General introduction
Chapter 1
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
Recent concern regarding cross-resistance of pathogens in humans have became
into the total ban of antibiotics as growth promotants in livestock in the European
Union on January 2006. Since the first restrictive measures were taken, and due to the
begining of the negative consequences of the ban, great efforts have been done to
look for alternatives or replacement strategies to maintain pig growth performance
and controlling enteric bacterial diseases. One of the ways that probably could help to
maintain productive indexes without the use of antibiotics would be those related
with the maintenance of a robust indigenous intestinal microbiota that helped the
animal to resist invasion by potentially disease-causing pathogenic bacteria.
Gastrointestinal microbiota is a complex and dynamic ecosystem that inhabits the
pig gut since birth, and have an important influence on the animal health: gut bacteria
provides essential products to the host, forms a key barrier against pathogens and also
plays important roles in gut morphology, immunity development, digestion and even
modulating gene host expression. However, our knowledge of this complex
ecosystem is still limited. Until recently, the major part of the studies of intestinal
microbiology have been based on traditional methods, although at present it is
recongnized that these methods misregard an important percentage of bacteria due to
failure of many of them to grow in a given culture medium. In this regard, the
development in the last years of high resolution molecular techniques based on 16S
ribosomal DNA gene has revolutioned our knowledge of complex microbial
populations such as the pig gut microbiota, since viability and later growth of cells is
not necessary to their study. Those studies have showed that the complexity of
microbial community is much greater than previosly thought. Among the diversity of
methods, quantitative PCR and some fingerprinting techniques like DGGE and tRFLP have been extensively used to study pig gut bacteria.
Recently numerous products have appeared in the market with the aim to maintain
production indexes of the antibiotics “age”. In this sense, different alternatives to
antibiotics growth promoters have been tested with promising results, although still
not comparable with those obtained with antibiotics. Among these new alternatives, it
may be remarcked the use of prebiotics, probiotics, organic acids, minerals at
pharmacological doses and plant extract mixtures. Most of them are thought to act
throught an effect on gut bacteria, by shifting the microbial equilibrium, whilst others
might be acting by other different mechanisms in the improvement of health and
2
INTRODUCTION
General introduction
INTRODUCTION
Chapter 1
performance. In this sense, further research is necessary to improve knowledge
regarding the mechanism of action of these compounds that undoubtly will help to
improve their proper use in field conditions. The use of molecular methods in
conjunction with traditional ones will be a key role in further studies regarding pig
gut microbiota.
3
TRIAL IV
TRIAL III
TRIAL II
TRIAL I
OBJECTIVES
4
LITERATURE
REVIEW
Chapter 2
TRIAL V
INTRODUCTION
Literature review
LITERATURE REVIEW
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
INTRODUCTION
Literature review
Introduction
Similarly to other microbial ecosystems, the establishment of the pig gut
LITERATURE
REVIEW
2.1. Development of the intestinal microbiota after birth
microbiota is a complex process that involves a first colonizing phase during which
followed by different
successional steps where diverse dominant groups become predominant. This process
continues as the pig matures, resulting finally in a characteristic and dynamic
bacterial community population for each individual (Rolfe et al., 1996; Zoetendal et
al., 2001). More than 500 different bacterial species of indigenous micro-organisms
OBJECTIVES
the gut of newborns is rapidly invaded by bacteria,
are usually described in the lower tract of the adult pig (van Kessel et al., 2004).
and occurrence over time, by multiplying at a rate that equals or exceeds their rate of
washout or elimination at an intestinal niche, or if not, by attachment to the gut wall
TRIAL I
To colonize the gastrointestinal tract, bacterial population need to be stable in size
to maintain a permanent colonization (Mackie et al., 1999). It is influenced also by
several factors of both bacterial and host origin. The main factors affecting the
availability and composition, the flow of digesta, pH, molecular oxygen and
oxidation/reduction potential (Stewart et al., 1993). Accordingly, pig microbiota
differs quantitatively and qualitatively throughout the gastrointestinal tract (Berg,
TRIAL II
colonization process are immune reactivity, the presence of gut receptors, nutrient
1996; Simpson et al., 1999), with the highest counts in the caecum and the colon.
There is also a horizontal stratification in the lumen, mucus lining and crypt spaces,
This chapter will focus on the pig gastrointestinal tract colonization from birth to
the adult age, paying special attention to the most recent microbiological works.
TRIAL III
with characteristic population in each section (Lee et al., 1984; Simpson et al., 1999).
At birth, the piglet gastrointestinal tract is sterile. However, from the moment the
fetal membranes are ruptured, the piglet is exposed to a huge variety of microbes. In a
TRIAL IV
2.1.1. First colonizers
short period of time, contact with the vagina, feces and skin of the mother, as well as
6
TRIAL V
with the environment starts the gastrointestinal colonization of the piglet’s gut
INTRODUCTION
Chapter 2
(Conway, 1997). Recently,
comparisons of bacteria metabolic fingerprintings
determined by Katouli and co-workers (1997) demonstrated that there was a high
LITERATURE
REVIEW
similarity among the flora of piglets and their dams during the early stages of the
animals life, confirming therefore that sows were the initial source of the gut flora for
piglets. In particular, the mother’s feces might be a key factor in this adquisition and
future microbiota development, as it is confirmed that piglets can consume up to 85 g
of feces per day (Sansom and Gleed 1981). However, in a few days, microbiota
OBJECTIVES
patterns change in the piglet and become more different from sow and characteristic
for each individual (Katouli et al., 1997).
The first bacteria detected in the piglet digestive tract are very diverse, reflecting
the miscellany of the microbial populations associated with the mother and the
environment (Ewing and Cole, 1994). However, in the following days, simplified
microbiota profiles have been characterized, which will become more complex with
TRIAL I
time, increasing its diversity as the animal grows (Conway, 1994; Favier et al., 2002;
Inoue et al., 2005). In this regard, may be remarked a comprehensive work by Swords
and co-workers (1993; Figure 2.1) who studied pig fecal microbiota evolution within
the first four months of life, and concluded that the establishment of the adult fecal
flora is a large and complex process with three different marked phases in the
TRIAL II
bacterial succession. The first phase corresponds with the first week of life, the
second one, from the end of the first week to conclusion of suckling, and the third
phase from weaning to final adaptation to dry food.
In this first phase, aerobes and facultative anaerobes from the sow and the
environment become the predominant bacterial groups, comprising 80% of the total
TRIAL III
flora by three hours after birth. The gut colonization is extremely fast; only twelve
hours after birth, total bacteria in distal colon reaches counts of 109 CFU/g colonic
content (Swords et al., 1993; Jensen et al., 1998).
First colonizers modify the gastrointestinal environment (by consumption of
molecular oxygen and reduction of the redox potential), making it more favorable for
TRIAL IV
the following colonization by anaerobes. Although not only the change in gut
environment is involved in the substitution of these first bacteria. Calostrum
immunoglobulins also act excluding antigens from entering the gut (Brandtzaeg,
2002). As a result, aerotolerant bacteria are gradually supplanted by strict anaerobes,
TRIAL V
7
INTRODUCTION
Literature review
and 48h after birth, piglets already show 90% of anaerobic bacteria (Swords et al.,
Figure 2.1 (A). Evolution of aerobic and anaerobic bacteria in piglet feces from
birth to 120 days of life (adapted from Swords et al., 1993).
LITERATURE
REVIEW
1993; Figure 2.1(A)).
120
ANAEROBIC
OBJECTIVES
AEROBIC
100
80
60
40
20
TRIAL I
120d
90d
60d
30d
25d
20d
15d
9d
10d
8d
7d
6d
5d
4d
3d
2d
24h
12h
9h
6h
3h
0h
0
Figure 2.1 (B). Total bacteria counts and percentage of coliforms, Bacteroides
spp. and Clostridium spp. in piglet feces from birth to 120 days of age (adapted from
Coliform bacteria, % Total bacteria
Bacteroides spp. , %Total bacteria
90
d
30
d
20
d
Clostridium spp. , %Total bacteria
8
TRIAL IV
90
d
30
d
20
d
10
d
8d
6d
4d
2d
12
h
6h
0h
90
d
30
d
20
d
10
d
8d
6d
4d
2d
12
h
6h
100
90
80
70
60
50
40
30
20
10
0
TRIAL V
100
90
80
70
60
50
40
30
20
10
0
0h
10
d
0h
90
d
30
d
20
d
10
d
8d
6d
4d
2d
12
h
6h
0h
0
8d
2
6d
4
4d
6
2d
8
12
h
10
6h
100
90
80
70
60
50
40
30
20
10
0
12
TRIAL III
Total bacteria, log10 UFC/g MF
TRIAL II
Swords et al., 1993).
INTRODUCTION
Chapter 2
Of these bacterial groups, lactobacilli and streptococci become the dominant
bacteria at the end of the first week of life and will be maintained for the whole
LITERATURE
REVIEW
suckling period with counts of around 107-109 CFU/g digesta (Swords et al., 1993;
Ewing and Cole, 1994).
Microbiota remains fairly stable in terms of species composition during the
second phase when the piglets receive milk from their mother (Drasar and Barrow,
1985; Mathew et al., 1998). The diversity of anaerobic bacteria increases in this
OBJECTIVES
period (Inoue et al., 2005) and supplantation of aerobic and facultative anerobic
bacteria by anerobic bacteria becomes almost completed in this phase. As has been
mentioned before, lactobacilli and streptococci continue being dominant bacteria,
which are well adapted to utilize substrate from the milk diet. Clostridium,
Bacteroides, bifidobacteria, and low densities of Eubacterium, Fusobacterium,
Propionibacterium and Streptococcus spp. are also usually found in this second phase
TRIAL I
(Radecki and Yokohama, 1991; Swords et al., 1993).
2.1.2. Weaning: the adaptation to dry food
Modern pig production involves very early and suddenly weaning, usually at
TRIAL II
three or four weeks of life. At this moment, the piglet is subjected to complex social
changes, including separation from its mother, separation from litter-mates and
exposure to unfamiliar counterparts, environmental and nutritional changes (Fraser et
al., 1998).
As a result, weaned piglets refrain from eating (Le Dividich and Herpin, 1994)
TRIAL III
and concurrently, profound changes in intestinal structure with associated disrupted
functional capacity take place (Hampson, 1986; Pluske et al., 1997) which lead to
growth stasis (McCracken et al., 1995, 1999; Figure 2.2).
In particular, anorexia leads to rapid changes in the microbiota as substrate
available for microbial fermentation depletes. As a consequence, during the first week
postweaning the microbiota becomes especially unstable, with a marked decrease in
TRIAL IV
biodiversity (Wallgren and Melin, 2001) which will be restored after a
reestablishment period of two or three weeks (Jensen et al., 1998). In this regard,
increases in biodiversity have been reported 24 days after weaning (Inoue et al., 2005;
Figure 2.3).
TRIAL V
9
INTRODUCTION
Literature review
Swords and co-workers (1993) defined weaning as the start of the third phase in
the main energy source instead of lipids, and more complex chemical composition as
the key factor in the microbiota change; major quantitative and qualitative changes
are described immediately after piglets are weaned (Mathew et al., 1996; Jensen,
LITERATURE
REVIEW
pig gut colonization process with the introduction of solid food with carbohydrates as
Figure 2.2. Review diagram of piglets post-weaning challenge.
WEANING
OBJECTIVES
1998; Konstantinov et al., 2004a).
• Nutritional Social Environmental
INMATURE DIGESTIVE
SYSTEM
INMATURE INMUNE
SYSTEM
STRESS
STRESS
TRIAL I
MULTIPLE CHALLENGES
FASTENING PERIOD
PERIOD
FASTENING
MICROBIOTAUNBALANCE
UNBALANCE
MICROBIOTA
VILLIHEIGHT
HEIGHT
VILLI
OPPORTUNISTIC
INFECTIONS
MALDIGESTION
MALABSORPTION
CRYPTDEPTH
DEPTH
CRYPT
POST WEANING DIARREA
POST-WEANING SYNDROME
GROWTH STASIS
TRIAL III
BACTERIALOVERGROWTH
OVERGROWTH
BACTERIAL
TRIAL II
MORPHOLOGICALCHANGES
CHANGES
MORPHOLOGICAL
There is a decrease in total culturable bacteria after weaning (Franklin et al.,
2002), with marked changes in some characteristics groups. Also, the third phase in
anaerobes by members of the gram-negative genus Bacteroides which will represent
one of the main bacteria populations in the adult pig (Swords et al., 1993). This
TRIAL IV
pig gut colonization is characterized by the supplantation of the gram-positive
agrees with Jensen (1998) who found that immediately after weaning, the main part
10
TRIAL V
of culturable bacteria from the large intestine were gram-negative. There is also
INTRODUCTION
Chapter 2
described a decrease in lactobacilli population parallel with an increase in
enterobacteria as a consequence of commercial weaning (Mathew et al., 1993, 1996;
LITERATURE
REVIEW
Jensen et al., 1998; Franklin et al., 2002). In fact, abrupt weaning has been associated
with a 100-fold drop in the numbers of lactobacilli in the intestine, and a 50-fold
increase in the numbers of Escherichia coli (Huis in’t Veld and Havennar, 1993).
The main result of this microbiota disruption in the period immediately following
weaning is that piglets become more susceptible to overgrowth with potentially
OBJECTIVES
disease-causing pathogenic bacteria (Hopwood and Hampson, 2003; Pluske et al.,
2003).
Figure 2.3. (A) Diversity, expressed as number of bands obtained by Temperature
Gradient Gel Electrophoresis (TGGE), of the intestinal microbiota in piglets from
birth to 14 days after weaning. (B) Dendogram based on TGEE profiles of one piglet.
Weaning takes places on day 25 (Inoue et al., 2005).
TRIAL I
A
TRIAL II
TRIAL III
B
TRIAL IV
TRIAL V
11
INTRODUCTION
Literature review
After weaning, the normal adult flora develops and, in the healthy adult animal, it
became stable and characteristic for each individual (Zoetendal et al., 1998; Simpson
et al., 2000). This adult microbial “climax” is influenced by environmental factors as
well as by host genotype with an increasing gradient of indigenous microbes from the
LITERATURE
REVIEW
2.1.3. Autochthonous microbiota in the adult pig
The stomach and small intestine contain relatively low numbers of bacteria
compared with the lower gastrointestinal tract (107-109 CFU/g fresh matter, in Jensen
and Jorgensen, 1994). The acidic conditions, the rapid flow of digesta and the rate of
bacterial washout restrict the bacterial population in these sections. However, the
OBJECTIVES
stomach to the cecum (Ewing and Cole, 1994).
ability of lactic acid bacteria to associate with the stratified squamous epithelial
of the stomach (pars oesophagea) allows their colonization, and this is
probably the reason why these bacteria become the predominant group in the upper
gastrointestinal tract (mainly lactobacilli and streptococci; Jensen, 2001). Beside
lactic acid bacteria, other groups like enterobacteria, Clostridium, Eubacterium and
TRIAL I
surface
Bifidobacterium are also found (Melin, 2001; Conway, 1994).
upper sections. The slower passage rate, the greater amount of digesta and a higher
pH result in an increased density and diversity of bacteria. This section of the
gastrointestinal tract is considered a transition zone preceding the large intestine
(Jensen
and
Jorgensen,
1994).
Lactobacillus,
Streptoccoci,
TRIAL II
In the distal small intestine, the environmental conditions slightly differ form the
Clostridium,
Enterobacteria, Bacillus and Bacteroides spp. are the most important culturable
The cecum and colon are the major sites for bacterial fermentation in the pig gut,
characterized by a high diverse population. The high amount of substrate, the slow
TRIAL III
bacteria described (Conway, 1994; Jensen, 2001; Hill et al., 2005).
digesta flow, the neutral pH and the low redox potential constitute the perfect
environment for the development of a diverse and stable microbiota (Fonti and Gouet,
Gaskins, 2003) with total counts of more than 1011-1012 CFU /g digesta (Ewing and
Cole, 1994). The majority of the culturable bacteria described in the pig cecum and
colon are gram-positive anaerobes: streptococci, lactobacilli, eubacteria, clostridia
TRIAL IV
1989). Several hundred anaerobic bacterial species coexist (Pryde et al., 1999;
12
TRIAL V
and peptostreptococci. The gram-negative bacteria cover only about 10% of total
INTRODUCTION
Chapter 2
culturable bacteria, most isolates belonging to the Bacteroides and Prevotella groups
(Russell, 1979; Salanitro et al., 1979; Moore et al., 1987; Table 2.1).
LITERATURE
REVIEW
Table 2.1. Main bacteria traditionally cultured from the pig gastrointestinal tract
(adapted from Stewart et al., 1999, in alphabetic order).
Bacteria
OBJECTIVES
Bacteroides (Prevotella) ruminicola
Bacteroides fragilis, B. suis, B. uniformis, B. furcosus, B. pyogenes, B. amylophilus
Bifidobacterium adolescentis, B. boum, B. suis, B. therophilum, B. pseudolongum
Butyribibrio sp., B. fibrisolvens
Clostridium sp., C. putrificum, C. welchii, C. perfringens
Enterococcus sp., E. avium, E. faecium, E. faecalis, E. hirae.
Escherichia coli and other members of the Enterobacteriaceace family
TRIAL I
Eubacterium sp., E. tenue, E. lentum, E. cylindroids, E. rectale
Fibrobacter succionogenes
Fusobacterium prausnitzii, F. necrophorum
Lactobacillus sp., L. acidophilus, L. brevis, L. crispatus, L. fermentum, L. johnsonii, L.
agilis, L. amylovorus, L. reuteri, L. plantarum, L. delbrueckii, L. salivarius
Megasphaera elsdenii
TRIAL II
Pediococcus halophilus
Peptostreptococcus anerobius
Propionibacterium acnes, P. granulosum
Ruminococcus sp., R. flavefaciens
Streptococcus sp., S. salivarius, S. bovis, S. morbillorum, S. intermedius, S. durans, S.
equines, S. intestinalis
TRIAL III
Recently, advances in molecular biology have greatly increased our knowledge of
this complex ecosystem. In particular, may be remarked an elegant work by Leser and
co-workers (2002; Table 2.2), who carried out an experiment where the pig
gastrointestinal microbiota was extensively described by 16S rDNA sequencing.
TRIAL IV
Surprisingly, they found that 83% of the sequences amplified were unknown
because had a <97% of similarity to any sequences in the database and therefore may
represent yet-uncharacterized bacterial genera or species. This confirms again the
high ignorance regarding microbial ecosystems that we still have today.
TRIAL V
13
INTRODUCTION
Literature review
Despite this high percentage of unknown bacteria, functional groups of bacteria
Table 2.2. Major phylogenetic lineages to which the phylotypes from the porcine
GI tracts were affiliated (adated from Leser et al., 2002).
Phylogenetic groupa
Eubacterium and relatives
Clostridium and relatives
Bacillus-Lactobacillus-Streptococcus
subdivision
Flexibacter-Cytophaga-Bacteroides group
Proteobacteria
Sporomusa and relatives
Mycoplasma and relatives
High-G+C bacteria
Spirochetes and relatives
Clostridium purinolyticum group
Planctomyces and relatives
Flexistipes sinusarabici assemblage
Anaerobaculum thermoterrenum group
a
No. of phylotypes
detected
125
109
Similarity (%)b
46
96.7
42
20
15
8
4
2
1
1
1
1
87.5
94.8
94.7
78.6
93.5
86.4
94.4
86.0
85.9
84.3
93.0
92.2
OBJECTIVES
which phylotypes were affiliated are shown.
TRIAL I
Bacteroides and Prevotella groups. In Table 2.2, the major phylogenetic lineages to
TRIAL II
the low-G+C gram-positive division (81%), and 11.2% were affiliated to the
LITERATURE
REVIEW
agreed to a great extent with culture results. The major phylotypes found belonged to
Phylogenetic grouping according to the Ribosomal Database Project.
Mean similarity of all the phylotypes affiliated to that group to the most closely related
sequences in the RDP alignment version 7.1.
TRIAL III
b
Summary
successional process that is influenced by several factors. It starts immediately after
birth, when environmental bacteria begin gut colonization. However, commercial
weaning, stresses the animal resulting in a disruption in the natural bacterial
TRIAL IV
The establishment of the pig gastrointestinal microbiota is a large and
succession with both quantitative and qualitative changes. In consequence, the pig
14
TRIAL V
becomes more susceptible to overgrowth with potentially disease-causing pathogenic
INTRODUCTION
Chapter 2
bacteria. After this alteration, the normal colonization continues and in the healthy
adult pig becomes a stable and characteristic ecosystem with Eubacterium,
LITERATURE
REVIEW
Clostridium and bacteria belonging to the Bacillus-Lactobacillus-Streptococcus
subdivision and the Cytophaga-Flexibacter-Bacteroides group as the main bacteria.
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
15
2.2. Main functions of the indigenous microbiota in the gut
Introduction
INTRODUCTION
LITERATURE
REVIEW
Literature review
The mammalian gut harbors a complex, dense, dynamic and spatially diversified
1000 different bacterial species colonize the adult intestine (Noverr and Huffnagle,
2004) with approximately 1014 bacteria (Pickard et al., 2004), ten fold higher than the
total mammalian cells (Van Kessel et al., 2004). This ecosystem is an active
OBJECTIVES
community of non-pathogenic micro-organisms. Studies suggest that between 500-
metabolic unit that provides essential products to the host, forms a key barrier against
pathogens and plays important roles in gut morphology (Coates et al., 1963),
even in modulating gene host expression (Hooper et al., 2001). These physiologic
contributions are reciprocated by the provision of stable niches in the intestine for the
TRIAL I
immunity development (Pabst et al., 1988), nutrient digestion (Wostmann, 1996) and
bacteria, making the relationship between the host and its microbiota a true
“mutualism” more than a “commensalism” as it has been traditionally described
This chapter focuses on the importance of the interaction between the microbiota
and the host, paying special attention to the role of bacteria on gut morphology, and
TRIAL II
(Darveau et al., 2003).
to the establishment of the gut barrier, nutrient digestion and immunity development.
Gut microbiota influences gut structure, function and maturation (Berg et al.,
1996; Falk et al., 1998). These effects are directly due to the presence of commensal
TRIAL III
2.2.1. Effects of indigenous bacteria on gut maturation and development
bacteria and have been largely studied by comparing germ-free animals bred and kept
in a sterile environment, with the same specie kept using conventional husbandry;
and biochemical changes, attributed to the microbiota, that have been called
microflora-associated characteristics (MACs; Midvedt, 1989; Box 2.1).
TRIAL IV
mainly mice and rats. Comparisons have demonstrated several anatomic, physiologic
Morphologic changes found in the absence of microbiota include: a reduction in
16
TRIAL V
intestinal mass per unit length, intestinal thickness and length (Wostmann, 1996), and
INTRODUCTION
Chapter 2
an enlarged caecum with a thinner mucosa. The reduction in intestinal mass can be
explained by a manifestly reduced cellularity of the lamina propia which contains
LITERATURE
REVIEW
fewer lymphocytes, plasma cells, and mononuclear cells (Van Kessel et al., 2004)
which may be related to the lack of microbiota stimuli on immune response. On the
other hand, the enlargement of the caecum is due to the accumulation of undegraded
mucus (Gustaffson et al., 1970). Carlstedt-Duke (1986) demonstrated that the
enlargement can be easily reversed by the monocolonization of germ-free rats with
OBJECTIVES
the mucolytic bacteria Peptostreptococcus micros.
At a histological level, the absence of microbiota is also related to thinner villi
and shorter crypts, and as a consequence villi:crypt ratio increased (Umesaki et al.,
1993, 1995; Wostmann, 1996). The shorter crypts are the reflection of a reduced
mitotic index and a cell turnover rate in the intestinal epithelium of germ-free animals
with a reduction in the number of cells (Alam et al., 1994). However, not all bacteria
TRIAL I
species exert the same effects on intestinal morphology. Recently, Lafuente and coworkers have demonstrated that whereas some commensal bacteria such as
Lactobacillus may improve the tightness of the barrier, other commensal-non
pathogenic species such as Escherichia coli may impair colonic barrier function and
increase the colonic permeability to luminal toxins (García-Lafuente et al., 2001). In
TRIAL II
spite of this, individual effects of isolated bacteria species on the intestinal epithelium
are probably not completely representative of the effect of these same bacteria in a
complex microbial ecosystem where the function of each individual is modulated by
the presence of the others.
The presence of bacteria in the gastrointestinal tract also affects its motility. In
TRIAL III
germ-free animals, the rate at which the digesta is moved by peristalsis along the
upper gastrointestinal tract is slower (Falk et al., 1998). Similarly to some other
characteristics usually described in germ-free animals, it has been seen that after
colonizing the animals with the normal caecal contents of a conventionally raised
animal, the motility is restored (Huseby et al., 1994). One possible cause of this effect
may be related with end-products of microbiota fermentation. Different research
TRIAL IV
groups have studied the effect of short-chain fatty acids on gut motility, including
systemic humoral and neural pathways as well as local reflexes and myogenic
responses (Yajima, 1985; Cherbut et al., 1996, 1997). Similarly, the presence of
lactobacilli, described as one of the main bacteria in the pig gastrointestinal tract
particularly in the gut upper sections (Hill et al., 2005), has also been related to the
TRIAL V
17
INTRODUCTION
Literature review
microbiota effect on gut motility. Moreover, in vitro studies have demonstrated that
(Tannock et al., 1999).
Box 2.1. Characteristics of germfree rodents compared with conventional rodents
LITERATURE
REVIEW
lactic acid (which is produced by these genera) is able to stimulate intestinal motility
Decreased intestinal motility
Decreased rate of villus epithelial cell renewal
Altered mucosal enzyme patterns
Increased oxigen levels
Decreased basal metabolic rate
Decreased cardiac output
Decreased regional blood flow
Decreased sintesis of vitamin K and vitamin B complex
No bile acid transformation in intestines
Lack of short-chain fatty acids
TRIAL I
PHYSIOLOGICAL/BIOCHEMICAL CHARACTERISTICS
TRIAL II
MORPHOLOGICAL CHARACTERISTICS
Increased cecum size
Decreased weight of intestinal wall
Decreased surface area
Thinner intestinal villi
Thinner lamina propia
Decreased size of liver, heart, adrenals…
Decreased blood volume
OBJECTIVES
with an indigenous microbiota (Berg et al., 1996).
2.2.2. Establishment of the gut barrier and colonization resistance
TRIAL IV
Decreased lymph node and spleen size
Decreased Peyer’s patches size
Decreased serum immunoglobulins levels
Decreased numbers of inmunoglobulin-A-producing lymphocites in lamina propia
Decreased number of intraepithelial T cells
Decreased imflammatory response
Delayed immune response against antigenic challenge
TRIAL III
IMMUNOLOGICAL CHARACTERISTICS
Besides the indigenous microbiota contribution to gut maturation and
18
TRIAL V
development, there is another direct effect that is essential for the protection of the
INTRODUCTION
Chapter 2
host against pathogenic invaders. The indigenous microbiota suppresses colonization
of incoming bacteria by a process named colonization resistance that is a first line of
LITERATURE
REVIEW
defense against invasion by exogenous, potential pathogenic organisms or indigenous
opportunists (Van der Waaij et al., 1989; Rolfe et al., 1996; Hooper et al., 1998). This
process involves several different complex interacting mechanisms of both the
bacteria and the host.
The host factors involved in colonization resistance are diverse: the peristaltic
OBJECTIVES
movement; the secretion of diverse digestive enzymes and electrolytes; the secretion
of mucus; epithelial cell desquamation; the gut associated lymphoid tissue; and
secretory IgA (Stewart et al., 1993). On the other hand, indigenous microbiota
prevents bacterial colonization by competing for epithelium receptors (Blomberg et
al., 1993; Bernet et al., 1994) and enteric nutrients (Stewart et al., 1999), producing
antimicrobial compounds such as bacteriocines (Brook, 1999) and metabolizing
TRIAL I
nutrients to create a restrictive environment which is generally unfavorable for the
growth of many enteric pathogens (Fons et al., 2000; Lievin et al., 2000).
Moreover, bacterial recognition and adhesion to receptors is not only a
prerequisite for colonization, which determines microbiota composition and
permanent colonization, especially in the upper gastrointestinal tract (Alander et al.,
TRIAL II
1999). It also determines antagonistic activity against enteropathogens (Coconnier et
al., 1993), modulation of the immune system (Schiffrin et al., 1997) and also the
improvement of healing in the damaged gastric mucosa (Elliot et al., 1998).
Several factors are involved in the control of bacterial attachment and thus in the
modulation of the indigenous microbiota profile (Freitas et al., 2002). Special interest
TRIAL III
is nowadays focused on genetic modulation of receptors by the host and the bacteria,
as we will see in the following chapters. Bacterial-mucose attachment appears
consequently as a key point defining indigenous microbiota composition and different
bacterial-mediated functions. Two main components are essential to the recognition
between the host and the bacteria: the glycoconjugates on the gut enterocytes and
TRIAL IV
bacterial adhesins.
TRIAL V
19
INTRODUCTION
Literature review
The gastrointestinal epithelium is covered by a layer of mucus, which forms a
barrier between the lumen content and the mucosa against chemical, microbiological
and physical injury (Forstner and Forstner, 1994). The presence of this mucus barrier
is also essential in the mechanisms of bacterial colonization and therefore in the
LITERATURE
REVIEW
2.2.2.1. Glycoconjugates of the mucosa as specific attachment site
Mucus is secreted by specialized epithelial cells called goblet cells and consists of
a continuous layer (100-200 µm in thickness, Pullan et al., 1994) overlaying the
epithelial surface (Specian and Oliver, 1991). The mucus is the result from noncovalent interactions between large and highly hydrated glycoconjugates that co-exist
OBJECTIVES
colonization resistance process (van Dijk, et al., 2002).
with other components such as water, peptides and surfactant phospholipids (Kindon
mucins are the key molecules in the bacterial recognition by the enterocytes, and
consist of a peptide core with many long side-chains of sugars (Mantle and Stewart,
1989). Different types of carbohydrate are involved: N-acetylglucosamine, galactose,
TRIAL I
et al., 1995; Matsuo et al., 1997). These high molecular weight glycoconjugates or
N-acetylgalactosamine, fucose, N-acetylneuraminic acid or sialic acid, mannose,
glucose and xylose. These glycoproteins are classified as either N- or Oof the lateral chain of asparagine whereas in O-glycoproteins the oligosaccharide is
attached to the oxygen atom of the lateral chain of serine or threonine (Mouricout and
TRIAL II
glycoproteins. In N-glycoproteins the oligosaccharide is attached to the nitrogen atom
Julien, 1987).
The
structural
diversity
of
these
carbohydrate
structures
on
mucin
recognition sites for adhesion of both commensal and pathogenic bacteria. When
indigenous bacteria are recognized by these receptors and occupy those, avoid the
TRIAL III
macromolecules and the different linking ways, becomes in a huge different target
attachment of newly incoming bacteria potentially pathogenics, retarding access of
microorganisms to mucosal surface (Forstner and Forstner, 1996).
establishment in the gut (Lu and Walker, 2001).
20
TRIAL V
carbohydrates enables some bacterial groups to colonize the mucus layer, favoring its
TRIAL IV
In addition to the colonization resistant effect, the ability to bind to mucin
INTRODUCTION
Chapter 2
Factors affecting gut glycoconjugates
LITERATURE
REVIEW
The epithelial cell receptors are host specific and are strongly affected by several
factors: genetics (Falk et al., 1998), cell maturity (Specian and Oliver, 1991), the
portion of the digestive tract involved (Barrow et al., 1980), the age of the host
(Dean, 1990; Turck et al., 1993), and the diet administered (Kotarski and Savage,
1979; Turck et al., 1993; Sharma and Shumacher, 1995). These factors result in
changes in susceptibility to colonization (King, 1995; Stewart et al.,1999).
OBJECTIVES
Different composition has been related to cell maturation. Immature goblet cells
produce mucins containing little sialic acid, and as they mature and migrate to the
villus tip, the sialic acid residues increase (Specian and Oliver, 1991). Age related
changes have also been found. A progressive change from α2,6 sialylation to α1,2
fucosylation of microvillar glycoconjugates occurs during postnatal development in
pigs (Kelly and King, 1991; King et al., 1993). Turck and colleagues (1993) found
TRIAL I
differences in fucose, glucosamine and sulphate contents of glycoconjugates when
comparing suckling with artificially fed piglets.
In recent years, special attention has been focused on the ability of microbiota to
modulate the expression of glycoconjugates by the host.
TRIAL II
Recent studies suggest that the host epithelial cell can express specific
glycoconjugates in response to the presence of bacteria (DDai, unpublished
observations, 2000; in Lu and Walker, 2001). Therefore, the gut microflora appears to
be the most responsible for: a) initiating production of host cellular glycoconjugates
needed for particular genera to join an intestinal niche (Umesaki et al., 1995; Freitas
et al., 2002), and b) to modulate the gut glycosylation pattern, both quantitatively and
TRIAL III
qualitatively by changing distribution of glycans (Freitas et al., 2002) and
consequently modifying potential sites for attachment.
This phenomena forms part of the “cross-talk process” that take place between the
host and its indigenous microbiota (Hooper and Gordon, 2001). Sharma and
Shumacher (1995) found that the presence of a determined microflora influences the
TRIAL IV
relative proportions of sulphated and sialylated types of mucins, and similarly some
recently investigations have demonstrated an exchange of biochemical signals, in the
form of soluble molecules, between Bacteroides thetaiotaomicron and the mice
enterocytes. This factor could cause alterations in fucosylated glycoconjugate
production through the induction of a host α1,2 fucosyltranseferase (Bry et al., 1996).
TRIAL V
21
INTRODUCTION
Literature review
These host-induced mucin modifications may potentially modify colonization of
used as an energy source by this bacteria. This could be a selective advantage when
competing with other bacteria for a niche with limited resources (Salyers et al., 1982).
In addition, it seems that bacteria have the ability to decide the best moment to start
LITERATURE
REVIEW
different bacteria, and also provide cellular fucosylated glycoconjugates that may be
host-induced modifications; Hooper and co-workers (1998) demonstrated that a
whether the intestinal environment has been adequately colonized before making the
energetic investment required for the initiation of this complex metabolic response
that involves modification of host properties.
OBJECTIVES
regulatory mechanism would allow different bacteria to achieve a consensus on
Besides modification of genetic expression glycoconjugates, mucins may also be
altered by bacterial endo- and exo- glycosidases. It has been described that some
a high quantity of glycoside hydrolases, that as a result, may degrade the
oligosaccharide chain of mucins. This leads to the creation or abolition of specific
TRIAL I
strains of Ruminococcus, Bifidobacterium, Clostridium and Peptostreptococcus hold
adhesion sites that may modify potential colonization, and also produces smaller
sugars that become available for other bacteria that are unable to digest the
2.2.2.2. Molecules involved in bacterial adhesion
TRIAL II
glycoconjugates by themselves (Hoskins et al., 1992; MacFarlane et al., 1999).
As described above, for the permanent colonization of the gut, the attachment of
indigenous bacteria to glycoconjugates is essential. However, although much effort
adherence of indigenous bacteria to the intestinal mucosa is not entirely known. The
interaction can be both specific by recognition of glycoproteins or glycoconjugates
TRIAL III
has been done to dilucidate the mechanisms of attachment to the intestinal wall, the
and inespecific involving complex mechanisms including bacterial motility,
chemotactic attraction, and non-specific attachment to the mucus gel (Kelly et al.,
Different bacterial surface elements are involved in the attachment of bacteria to
the mucopolysaccharides. These elements include molecules of protein type, such as
TRIAL IV
2005).
outer membrane proteins and fimbriae (pili) which are described in the major part of
22
TRIAL V
gram negative bacteria (Costerton et al., 1981). Other fimbrial structures and
INTRODUCTION
Chapter 2
fimbriosomes have been described in some hyperadhesive strains (Abraham et al.,
1985). Specifically, mannose sensitive fimbriae, also called type-1 fimbriae, which
LITERATURE
REVIEW
are associated with several bacteria from the Enterobacteriaceae family, including
Esherichia, Klebsiella, Shigella and Salmonella spp. (Knutton et al., 1985, 1987;
Clegg and Gerlach, 1987). Other fibrilar structures have also been described in
enterotoxigenic E. coli K88 (Bijlsma and Bouw, 1987).
Some other bacteria present non-protein type adhesines. Most are polysaccharides
OBJECTIVES
of the capsule or slime as lipotechoic acids that are mainly present in gram positive
bacteria (Sato et al., 1982; Contrepois et al., 1988). The surface of gram-positive
bacteria such as bifidobacteria, streptococci and staphylococci presents a linear
polyglycer-phosphate anchored to the cytoplasmic membrane which is known as
lipoteichoic acid (Poxton and Arbuthnott, 1990; Kelly et al., 1994).
TRIAL I
2.2.3. Effects of indigenous microbiota on immune response
It is well known that the resident microbiota also affects immunity in the host,
since it is usually described as the major source of antigenic material for the animal. It
is especially important in early life when the immune system is still not completely
TRIAL II
developed, particularly in piglets, that are born with the immune system immature.
Newborn piglets depend completely on the passive transfer of maternal antibodies by
calostrum and milk and do not develop an active immunity until 4-7 weeks of age
(Stokes et al., 1992; Gaskins and Kelley, 1995).
Considering this fact and that commercially reared piglets are early weaned at 3-5
TRIAL III
weeks, the knowledge of the host-bacteria cross-talk in relation to the host immune
system acquires special importance. This bacterial stimulus is especially important in
early life, in order to prime the immune system in the correct way and for the whole
life life and to maintain a functional immune system (Kelly and King, 2001).
Studies comparing germ-free and conventional-reared animals have demonstrated
that the presence of bacteria in the gastrointestinal tract strongly influences the
TRIAL IV
maturation and development of local and systemic immunity (Cebra et al., 1999).
Particularly, commensal bacteria play a key role in the development of the gut
associated immune system (Fioramonti et al., 2003). In the absence of microbiota, the
animal mucosal-associated lymphoid tissue is underdeveloped, with defects of cell-
TRIAL V
23
INTRODUCTION
Literature review
mediated immunity (MacDonald and Carter, 1979). The numbers of lymphocytes in
and lymph nodes also decrease in size (Umesaki et al., 1993; Wostmann, 1996);
macrophage chemotaxis and phagocytic activity are inhibited (Starling and Balish,
1981); and the immunoglobulin class profile is also altered, with much lower
LITERATURE
REVIEW
the lamina propia are decreased; and intestinal lymphoid aggregates, Peyer patches
concentration of IgG and low or no production of IgA (Wostmann et al., 1996).
exact mechanisms by which microbial colonization modulates the immune system are
still largely unknown (Cebra et al., 1999); even though it is thought that they involve
complex events that are probably triggered following the route of antigen uptake and
OBJECTIVES
Despite these marked effects of indigenous bacteria on immune response, the
processing (Kelly and King, 1994).
over-react against indigenous bacteria, and becomes “tolerant” is not completely
known. It seems clear that the immediate acquirement of microbiota during the postnatal period is essential for the development of tolerance to indigenous bacteria and
TRIAL I
In a similar way, the precise mechanism by which the immune system does not
also to other luminal antigens. The presence of pattern recognition receptors in
immune and epithelial gut cells may be behind this adaptative process (Hooper et al.,
2.2.3.1. Commensal bacteria tolerance-ignorance
TRIAL II
2001; Shanahan, 2002).
Dilucidation of how the commensal bacteria tolerance is established, retaining the
resident bacteria on immune system development. Although much effort is focused
on this field, it is still not clear the exact mechanisms involved.
There are several host factors in a healthy immune system that control bacterial
TRIAL III
capacity to respond to pathogens seems the key to clarify the described effects of
community within the gut lumen and gut wall: the mucus layer described above; the
interfollicular populations of T cells; and the gut lamina propia that presents a broad
spectrum of lymphoid cells, especially IgA plasmablasts, T cells, and dendritic cells,
and the intraepithelial leukocytes (Cebra et al., 1999; Stokes et al., 2001). The role of
TRIAL IV
Peyer patches; organized lymphoid tissues that contain B lymphoid follicles and
the secretory IgA which promotes bacterial niches formation but also limits the
24
TRIAL V
expansion and translocation of pathogens is especially important (Bollinger et al.,
INTRODUCTION
Chapter 2
2003; Suzuki et al., 2004) as are the antimicrobial peptides produced by Paneth cells
in the intestinal crypts (Ayabe et al., 2004).
LITERATURE
REVIEW
Among these host factors, recognition of bacteria by immune and epithelial gut
cells seems to be the key in the tolerance-establishment process. It relies on a wide
repertoire of specific receptors, which recognize highly conserved structures of
microorganisms (pathogen-associated molecular patterns; PAMPs) called pattern
recognition receptors (PRRs, Kopp and Medzhitov, 1999).
OBJECTIVES
The nucleotide-binding oligomerization domain molecules (NODs) and Toll-like
receptors (TLRs) are pattern recognition receptors. There are several examples of
TLRs that have been described to respond to bacterial stimulus: lipotheichoic acid
and peptidoglycan of gram positive bacteria are recognized by TLR2;
lipopolysaccharides of gram negative bacteria are recognized by TLR4; and flagellins
and bacterial DNA are recognized by TLR5 and TLR9 respectively. When a pathogen
TRIAL I
is recognized it results in signaling of immune cells (Akira et al., 2001; Schiffrin and
Blum, 2002), and immediately starts the synthesis of antimicrobial peptides,
cytokines and chemokines, and dendritic cells are also activated to eliminate it (Netea
et al., 2004, Kelly et al., 2005). In this regard, it has been recently demonstrated that
TLRs are coupled to signal transduction pathways that control expression of a variety
TRIAL II
of inducible immune-response genes (Kopp and Medzhintov, 1999).
However, whereas the immune system reacts against potential pathogens
recognized by the different TLRs, evidence suggests that when an indigenous bacteria
is recognized by a specific receptor, there is a tolerance against commensal
microbiota without signaling the immune system to eliminate it. Therefore, it seems
TRIAL III
that this bacterial recognition by TLRs may be the key in the host ability to
discriminate between pathogen and commensal bacteria. Although the exact
mechanism is not known yet, different hypothesis have been proposed to clarify the
tolerance process. One explains indigenous tolerance by the presence of difference
traits (PAMPs) in commensal bacteria that might be absent in pathogens (Schiffrin
TRIAL IV
and Blum, 2002). Matzinger (1998) postulated that in addition to this first recognition
by TLRs, that would be common to pathogens and commensal bacteria, a second
signal might also be included to initiate the appropriate response to pathogenic
bacteria. Other studies suggest that lymphocytes may downregulate pro-imflamatory
responses by intestinal epithelial cells to commensal bacteria (Haller et al., 2002) and
TRIAL V
25
INTRODUCTION
Literature review
recently, Kelly and co-workers (2005) suggested that the absence of some of the
contribute to the tolerance of the gut towards its microbiota.
However, it is important to remark that, in contrast with this local immune
tolerance against commensal bacteria described above, Macpherson and co-workers
LITERATURE
REVIEW
members of the TLRs family on the apical surfaces of epithelial cells might
(2005) demonstrated recently that the host systemic immune system remains naïve to
bacteria leaves the gut. They found that pathogen-free mice did not have specific IgG
against Enterobacter cloacae (a dominant member of its commensal flora) but it was
induced after intravenous injection of live micro-organisms. By this way, the host
OBJECTIVES
resident bacteria. It results in an effective immune response when an indigenous
preserves the ability to mount an effective systemic response against commensal
epitopes when necessary. It can be achieved by the compartmentalization that the
travel only from Peyer’s patches to the mesenteric lymph nodes without re-circulation
within the body (Macpherson et al., 2005; Figure 2.4).
TRIAL I
immune system has. When dendritic cells picks up commensal bacteria, they can
The microbiota plays a very important role in the digestion of the dietary
compounds that are not degradable by the pig endogenous enzymes, especially in the
large intestine, where materials are retained for prolonged periods of time. Therefore,
TRIAL II
2.2.4. The role of microbiota on digestion and absorption of nutrients
bacterial interaction with the host differs in the upper and lower gastrointestinal tract.
Whereas in the proximal gut, bacterial competition for absorbable nutrients could be
dietary residue that reach the distal gut (mainly carbohydrate polymers) is beneficial
to the host because it extracts nutrient value from otherwise poorly utilized dietary
TRIAL III
more detrimental than beneficial to the host, microbial digestion of non-digestible
substrates. Studies comparing conventional with gnotobiotic animals have proved that
in germ-free animals, while utilization of polysaccharides is less complete, the
1999).
26
TRIAL V
absorption seems to be affected (Fuller and Reeds, 1998, and reviewed by Tannock,
TRIAL IV
utilization of dietary lipid is more efficient. Also, amino acid absorption and mineral
INTRODUCTION
Chapter 2
Figure 2.4. Intestinal immune geography of responses to commensal bacteria.
Commensal bacteria are largely restricted from gaining access due to the physical
LITERATURE
REVIEW
epithelial and mucus barriers (Macpherson et al., 2005).
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
27
INTRODUCTION
Literature review
Carbohydrates are the main energy substrate for bacteria, as a result of the
inability of mammals to produce enzymes capable of degrading dietary fiber (the sum
of polysaccharides and lignin and resistant starch which are not digested by the
endogenous secretions of the gastrointestinal tract (Trowell et al., 1976; Englyst,
LITERATURE
REVIEW
2.2.4.1. Carbohydrate utilization by indigenous bacteria
1989). The digestion of those compounds depends totally on the activity of different
and xylanases (Salyers et al., 1977; Varel and Yen, 1997).
Pig microbiota harbors different highly cellulolytic and hemicellulolytic bacterial
species as Fibrobacter succinogenes, Fibrobacter intestinalis, Ruminococcus albus,
OBJECTIVES
bacteria that produce saccharolytic enzymes, cellulases, hemicellulases, pectinases
Ruminococcus flavefaciens, Butyrivibrio spp., and Prevotella ruminicola (Varel and
preferently fermented by lactobacilli (Graham et al., 1986) which are specially
important in the hindgut (Hill et al., 2005).
TRIAL I
Yen, 1997). Other carbohydrate substrates such as B-glucans and pectins are
The fermentation of carbohydrates in the pig colon results in the production of
high SCFA concentrations (70 to 100 mM) , lactic acid and gases (hydrogen, carbon
proportions depending of the gastrointestinal section. Whereas lactic acid is the main
organic acid in the stomach and small intestine, SCFA predominate in the colon and
cecum. A typical ratio of 60 acetate: 25 propionate: 15 butyrate is described in the
TRIAL II
dioxide, methane) (Bach Knudsen et al., 1991) varying in concentration and relative
lower pig gastrointestinal tract (Bach Knudsen et al., 1991).
Short chain fatty acids production is affected by several factors such as: the type
gastrointestinal tract transit time and microbiota (Allison and MacFarlane, 1989;
Wang et al., 2004).
TRIAL III
and chemical structure of polysaccharides; substrate redox and availability; and
SCFA are rapidly absorbed from the gut lumen (Argenzio and Southworth, 1974).
Acetate reaches the systemic circulation and acts as an energy substrate for muscle
liver (Montagne et al., 2003). Of special interest is butyrate which is the main energy
substrate for colonocytes and promotes a normal phenotype in these cells. SCFA
TRIAL IV
tissue (Cummings and Englyst, 1987), and propionate is converted to glucose in the
production is related to a normal large bowel function and to prevention of pathology,
28
TRIAL V
through its action in the lumen and on the colonic musculature and vasculature
INTRODUCTION
Chapter 2
(Topping and Clifton, 2001). Their contribution to maintenance energy requirement
has been estimated as 15 to 24 % in finishing pigs (Dierick et al., 1989; Yen et al.,
LITERATURE
REVIEW
1991; McBurney and Sauer, 1993).
Besides the contribution of SCFA to energy pig requirements and colonocytes
nutrition, other implications in animal health have been described. Diarrhea is limited
as SCFA stimulate the reabsorption of water and sodium (Roediger and Moore,
1981), and because, especially in acidic conditions, high contentrations of SCFA have
OBJECTIVES
been inhibit the growth of certain opportunistic pathogens as Salmonella, Clostridium
difficile and Escherichia coli growth is inhibited by SCFA (Prohaszka, 1986; May et
al., 1994).
2.2.4.2. Protein utilization by indigenous bacteria
TRIAL I
Microbiota can use nitrogen from dietary nitrogenous compounds as well as
enzymatic secretions of the host, mucin, and sloughed epithelial cells (Yen, 2001).
Bacteria also have the ability to utilize N not only in the form of protein but also from
other organic or inorganic sources. In particular, urea coming from plasma can be
efficiently utilized by bacteria for the synthesis of their own proteins; this is
TRIAL II
confirmed by the high amount of urea found in the colon of germ-free rats (Moreau et
al., 1976; Forsythe and Parker, 1985). Degradation of protein by bacteria in the small
intestine seems to be scarce. Salter (1984) found a similar degree of protein digestion
at the end of the small intestine of pigs reared in germ-free conditions compared with
conventional pigs, although other authors have found some amino acid degradation
produced by upper gastrointestinal tract bacteria that would diminish their availability
TRIAL III
to the pig (Gaskins et al., 2001). In the large intestine proteolytic fermentation is very
important in contrast to small intestine. As carbohydrate sources become depleted due
to fermentation by bacteria, the fermentation changes and becomes more proteolytic
(Piva et al., 1995).
In the large intestine numerous bacterial species may use peptides and aminoacids
TRIAL IV
as a source of carbon, nitrogen and energy. As a result, branched-chain VFAs are
formed by the use of branched chain amino acids valine, leucine and isoleucine
(MacFarlane et al., 1992).
TRIAL V
29
INTRODUCTION
Literature review
However, this proteolytic fermentation can also lead to the formation of
1983; MacFarlane et al., 1992; Williams et al., 2001).Bacteria belonging to the genera
Bacteroides, Clostridium, Enterobacterium, Lactobacillus and Streptococcus possess
the ability to produce amines by decarboxilation of amino acids (MacFarlane and
LITERATURE
REVIEW
potentially toxic metabolites such as NH3, amines, phenols and indols (Russell et al.,
MacFarlane, 1995). Aromatic aminoacids are metabolized into phenols and indols
Bifidobacterium genera (MacFarlane and MacFarlane, 1995).
A wide range of intestinal bacteria possess urease activity although studies have
not been conducted on the pig. An excess in urease activity compared to the ability of
OBJECTIVES
compounds, mainly by bacteria from Bacteroides, Lactobacillus, Clostridium and
bacteria to synthesize new protein can lead to an increase in ammonia. Ammonia
production has been related to an impaired development of the mucosa of the
TRIAL I
intestine, with a reduced villus height, and may also affect pig metabolism thus
reducing animal performance (Visek, 1984; Nousiainen, 1991).
2.2.4.3. Lipid utilization by indigenous bacteria
digestion, little attention has been given to the digestion and metabolism of lipids by
the commensal bacteria.
TRIAL II
Despite the high body of evidence of microbial collaboration in carbohydrate
Especially important is the metabolism of bile acids produced by the intestinal
microbiota. Microbial deconjugation and dehydroxylation of bile acids impair lipid
as certain secondary bile acids are citotoxic and potentially carcinogenic (Baron and
Hylemon, 1997). It has been found that E. coli, Bacillus cereus, Streptococcus
faecalis, Bacteroides spp., Eubacterium spp., and Clostridium spp. have the ability to
dehydroxilate bile acids.
TRIAL III
absorption by the host animal (Eyssen, 1973) and produce toxic degradation products,
Studies with germ-free mice have demonstrated that
lactobacilli contribute at least 74% of the total conjugated bile acid hydrolase activity
Several
bacteria
such
as
Clostridium
spp,
Eubacterium
lentum,
Peptostreptococcus spp. and Ruminococcus spp. also have different dehydrogenases
TRIAL IV
(Tannock et al., 1989).
capable of bile acid transformation. Advantages of metabolising this substrate may
30
TRIAL V
rely on the energy obtained from bile acid transformation, but more probably on the
INTRODUCTION
Chapter 2
growth inhibition of competing bacteria due to the toxicity of some of the compounds
released (Baron and Hylemon, 1997). Deconjugation of bile acids can affect
LITERATURE
REVIEW
negatively the digestion of dietary fatty acids as they act as emulsifiers, facilitating
their process of absorption. In this regard, digestion of lipids in gnotobiotics rats has
been found to be higher that in normally reared animals (Fuller and Reeds, 1998,
reviewed by Tannock, 1999).
Moreover, microbiota increases biohydrogenation of unsaturated fatty acids,
OBJECTIVES
resulting in a relative high proportion of stearic acid that is less well absorbed (Yen,
et al., 1991). Cholesterol, dietary sterols, and other lipids are also altered by
microbiota in the large intestine (Ratcliffe, 1991). Cholesterol is reduced to
coprostanol and coprostanone by the microbiota. Germ free rats excrete unmodified
cholesterol whereas conventional reared rats excrete coprostanol and coprostanone in
amounts of up to 55% of the total fecal sterols (McNamara et al., 1981). Different
TRIAL I
bacteria belonging to the Eubacterium genus such as Bacteroides, Bifidobacterium
and Clostridium possess the ability to metabolize cholesterol to coprostanol (Baron
and Hylemon, 1997).
Summary
TRIAL II
Mammals have co-habited with gut bacteria during thousand of years. This coevolution has become a narrow relationship with an established balance between the
eukaryotic and prokaryotic cells. Different studies have demonstrated that a
continuous cross-talk exists between them. It results in several beneficial effects for
both, becoming in a mutualist association. Bacteria achieve steady niches in the gut,
TRIAL III
with a stable environment and nutrient afford which is reciprocated to mammalian
host that obtains several benefits such as protection (immune system development
and homeostasis, the barrier effect), trophic (gut evolution and maturation) and
nutritional effects.
TRIAL IV
TRIAL V
31
INTRODUCTION
Literature review
Introduction
Since the 1940’s, when antibiotics were first used as growth promoters,
LITERATURE
REVIEW
2.3. Modulation of intestinal equilibrium through the feed
commercial pig diets have been regularly fortified with antibiotics in prophylactic
efficiency (Cromwell, 2002; Mroz, 2003). Currently it is known that the efficacy of
growth promotant antibiotics is mainly due to modification of the microbial
ecosystem and to subsequent direct and indirect effects on the host animal. However,
antibiotic specificity for microbial populations differ, and neither their effects on
OBJECTIVES
doses to prevent gastrointestinal disorders and to improve growth rate and feed
specific bacterial populations nor their exact mode of action promoting animal growth
Recent concerns regarding cross-resistance of pathogens in human therapy
(Hillman, 2001) have led to the total withdrawal of antibiotics as growth promoters in
TRIAL I
are completely defined (Gaskins et al., 2002).
the European Union. The first consequences of the ban are appearing already, with
lower post-weaning daily weight gain and a higher prevalence in post weaning
restrictive actions by the European Union were carried out, huge efforts are being
made to seek alternative or replacement strategies for controlling enteric bacterial
diseases by the maintenance of the piglet gastrointestinal ecosystem.
TRIAL II
diarrhea (Casewell et al., 2003). In the light of these results, and since the first
This chapter will focus on the major feed strategies that currently are used to
manage the pig gastrointestinal ecosystem with special attention to those used in
TRIAL III
young pigs.
2.3.1. Macro-ingredients
The main source of growth substrate for the gastrointestinal microbiota comes
ecosystem may be the modification of the amount and type of substrate available for
its use. This allows a direct and simple control over the process of fermentation in the
TRIAL IV
from the diet; thus, the single and most important control for the bacterial gut
gastrointestinal tract through pig feed composition (Jensen et al., 2003) that produces
32
TRIAL V
changes in microbiota and in the dominant bacteria inhabiting the gastrointestinal
INTRODUCTION
Chapter 2
tract (Conway, 1994). Specifically simple sugars are the main growth substrates to
bacteria in the upper gastrointestinal tract, whilst in the large intestine, where the
LITERATURE
REVIEW
major biomass is located, dietary fiber is the major substrate for pig gut microbiota
(Bach Knudsen et al., 1991; Hampson et al., 2001).
2.3.1.1. The role of dietary fiber
OBJECTIVES
Dietary fiber was first defined by Trowell et al. (1976) as “the sum of lignin and
polysaccharides that are not digested by endogenous secretions of the digestive tract
in the man”. The concept is applied also in monogastric animals. It consist of mainly
non-starch polysaccharides (cellulose, hemicellulose, xylans, beta-glucans, fructans,
mannans, pectins) resistant starch and lignin (Conway, 1994; Bach Knudsen, 2001).
Although non-digestible oligosaccharides can also be included in the dietary fiber
TRIAL I
definition, they will be exposed in the prebiotics chapter as usually are classified in
this group (Gibson and Roberfroid, 1995).
Starch and NSP (non-starch polysaccharides) are the main plant polysaccharides.
Pig lacks of endogenous enzymes capable of degrading NSP and, although amylose
and amylopectine from starch are susceptible to be hydrolysed by pig gastrointestinal
TRIAL II
enzymes, usually these compounds do not reach complete hydrolysis. The part of this
starch that is not digested, named resistant starch, together with NSP reach the lower
gastrointestinal tract where are susceptible to bacterial fermentation (Montagne et al.,
2003). Starch may be resistant to enzymatic hydrolysis for three reasons that have
determined its classification into three main types: RS1 includes resistant starch
trapped within whole plant cells and food matrices, thus physically inaccessible to the
TRIAL III
enzymatic host package; RS2 comprises poorly gelatinised starch granules that are
highly resistant to digestion by α-amylase; and RS3 comprises retrograded starch
(Englyst et al., 1992).
Different factors influence the response of microbial fermentation to fiber
administered in the diet. The most important are the source of dietary fiber (its
TRIAL IV
solubility, degree of lignification and processing) and the level of inclusion in the diet
(Bach Knudsen and Hanse, 1991; Macfarlane and Cummings, 1991, Jensen, 1998).
Although the ability of dietary fiber to modulate the gastrointestinal microbiota
has been clearly demonstrated, there is still a lack of knowledge about the specific
TRIAL V
33
INTRODUCTION
Literature review
effect of different types and amounts of fibre on particular microbial groups and
indigenous microbiota. Different authors have studied the influence of different types
and doses of fiber in the pig diet, denoting changes in the composition and metabolic
activity of the large intestinal microbiota in pigs.
LITERATURE
REVIEW
about its rational use to promote the establishment of a gut health promoting
Several works have shown how increases in fiber content in the diet can modify
this regard, Bach Knudsen and co-workers (1991) in a trial where diets with different
sources and levels of wheat and oat dietary fibre were administered, found marked
changes in total microbial activity throughout the pig gastrointestinal tract. In
OBJECTIVES
total microbial load and total bacterial activity throughout the gastrointestinal tract. In
agreement, Jensen and Jorgensen (1994), when administering a high–fiber diet (based
on barley supplemented with pea fiber and pectin) to adult pigs, found an increase in
activity in all segments of the hindgut. Modulation of microbiota activity has been
confirmed by other authors. Varel and Yen (1997) found that the administration of a
TRIAL I
the amount of total culturable bacteria in the stomach, and a higher total microbial
high-fiber diet increases total bacteria activity, as demonstrated by the 5 times greater
ATP quantity, and 5 to 9 times CO2 and CH4 produced in the gastrointestinal tract of
In a similar study from our group (Morales et al., 2002),we found that
administration of diets rich in maize or sorghum and acorn produces differences in
bacterial enzymatic activities in the lower gastrointestinal tract; once more showing
ability of microbiota to adapt to substrates offered. Recently, Martinez-Puig and co-
TRIAL II
pigs.
workers (2003) have also found increases in total bacteria activity, measured as
growing pigs, compared to corn starch. Pigs fed the potato starch diet also showed a
greater SCFA concentration in the hindgut than pigs fed corn diet.
In addition to changes in total bacteria loads and activity, the ability of microbiota
TRIAL III
purine bases content, when potato starch (highly resistant) was administered to
to adapt by changing in species composition has also been shown. The swine gut
harbors highly active ruminal cellulolytic and hemicellulolytic bacterial species
genus), Ruminococcus flavefaciens, R. albus, Butyrivibrio and Prevotella
ruminicola], which indicates the high potential that pigs have to profit from dietary
TRIAL IV
[Bacteroides succinogenes, B. intestinalis (currently re-classified into the Fibrobacter
fiber by microbiota utilization (Varel et al., 1982, 1985). In response to an increase in
34
TRIAL V
dietary fiber, the microbial ecosystem is able to adapt by increasing total cellulolitic
INTRODUCTION
Chapter 2
populations (Varel and Pond, 1985; Table 2.3). Moreover, when adult sows were fed
with a high-fiber diet (35% dehydrated alfalfa meal) changes in particular species
LITERATURE
REVIEW
were found, with increased numbers of Ruminococcus and Bacteroides compared to
animals receiving a low-fiber diet (based on corn and soybeans; Varel et al., 1984).
Table 2.3. Number of cellulolytic bacteria from fecal samples of sows fed diets
containing various levels of fiber (Varel and Pond, 1985).
OBJECTIVES
Cellulolytic bacteria (x 108 CFU /g dry matter)
Days on diet
TRIAL I
0
5
14
21
35
49
70
98
Overall
a, b
Control
20% corn
40% alfalfa
96% alfalfa
14.7
10.1
22.4
28.4
27.8
24.6
25.0
33.3
23.3b
6.0
10.2
17.5
16.9
16.3
32.8
9.3
12.5
15.2b
10.8
34.4
18.8
41.3
105.3
43.5
56.5
50.2
45.1a
14.1
56.5
24.2
71.0
54.9
76.3
59.3
63.7
52.5a
Means with different superscripts differ (p< 0.05)
TRIAL II
The administration of different vegetal sources of starch (more or less resistant)
has also been related with specific changes in bacteria species. When potato starch,
corn and waxy corn were administered to young pigs, shifts in the gastrointestinal
ecosystem were found, as was recently demonstrated by MacFarland (1998; Table
TRIAL III
2.4). Animals fed with potato starch had significantly lower coliform and E. coli
population in relation to the other two starches. Moreover, combinations of these
starches produced intermediate effects in comparison with individual starches,
suggesting that this form of manipulation should have the potential to accurately
control microbial population within the gut.
TRIAL IV
Direct modification of starches as amylose/amylopectine ratio and retrogradation
has also shown potential to modify the gastrointestinal ecosystem composition. Reid
and Hillman (1999) found marked decreases in total anaerobic counts when diets
were supplemented with a high amylopectine content starch, and increases in
TRIAL V
35
INTRODUCTION
Literature review
Lactobacillus spp., when animals were fed a diet rich in retrogradation of
starch.
Moreover,
retrogradation
decreases
the
coliform
population, which was reflected in a high lactobacilli:coliform ratio in distal colon,
specially with the high amylopectine starch.
In a similar way, a recent work of Martínez-Puig and co-workers (2006)
LITERATURE
REVIEW
amylopectine-rich
demonstrated marked different microbial patterns in hingut digesta after feeding
microbiota depending on the diet administered.
Table 2.4. Selected bacterial counts (CFU / g wet weight) in the proximal colon
OBJECTIVES
growing pigs with potato or maize starch. T-RFLP profiles showed different
of weaned piglets fed meal diets containing different source of starch (MacFarland,
a, b
Waxy
Corn
Corn
9.82a
1.90ª
1.73ª
9.62a
5.20a
7.16a
8.66b
6.93b
6.62bc
8.62b
6.44ab
7.43ab
8.88b
7.88b
7.39c
8.76ab
7.43b
7.16a
Potato/
Waxy
Corn
Potato/
Corn
Corn/
Waxy
Corn
10.05a
6.12b
5.99bc
9.67a
6.11ab
8.27bc
9.86a
4.24ab
3.64ab
9.67a
4.69a
8.54c
9.56ab
7.12b
6.52bc
9.17ab
6.19ab
7.17a
TRIAL II
Total anaerobes
Coliform bacteria
Escherichia coli
Lactobacillus spp.
Enterococcus spp.
Bacteroides spp
Potato
TRIAL I
1998, reviewed by Hillman, 2001).
Means with different superscripts differ (p< 0.05).
different types and amount of fiber added into the pig diet, today consensus regarding
its inclusion in the diet, particularly of young animals, does not exist. This lack of
TRIAL III
Although potential benefits on gastrointestinal microbiota can be related to
consensus is due to controversial results regarding inclusion of different sources of
dietary fiber in the diet and the occurrence of gastrointestinal disorders such as postmain intestinal disorder in the immediate post-weaning period, and although
multifactorial, it is associated with proliferation of enterotoxigenic haemolytic E. coli
in the small intestine. In growing pigs, swine dysentery is one of the most important
TRIAL IV
weaning colibacillosis and swine dysentery. Post-weaning colibacillosis (PWC) is the
diseases. It is caused by Brachyspira hyodysenteriae, which produces colitis in the
36
TRIAL V
lower gastrointestinal tract (Pluske et al., 2002). A few years ago, it was suggested
INTRODUCTION
Chapter 2
that the administration of fiber from oats, wheat, and barley supports protection
against proliferation of enteropathogen E. coli and the occurrence of PWC in piglets
LITERATURE
REVIEW
(Thomlinson and Lawrence, 1981) and also the insoluble fiber limited the severity of
PWC (Bertschinger and Effenberger, 1978). However, recent studies suggest that
diets high in fermentable carbohydrate sources, such as soluble NSP in weaner diets,
are detrimental to post-weaning growth and also have a positive correlation postweaning colibacillosis occurence. McDonald and co-workers (1997, 1999, 2001)
OBJECTIVES
reported an increased intestinal proliferation of E. coli in piglets infected
experimentally when they were fed fiber enriched diets (guar gum and pearl barley),
and in non-infected piglets when they were fed carboxymethylcellulose.
Similarly, the effect of dietary fiber on swine dysentery is also controversial.
Different works have reported that diets low in dietary fiber and resistant starch
prevented pigs from infection with Brachispira hyodisenteriae, and thus from swine
TRIAL I
dysentery disease (Pluske et al., 1996a; Durmic et al., 1998). However, recently
works from Kirkwood and co-workers (2000) and Lindecrona and co-workers (2003)
did not confirm these results, postulating that inclusion of fiber in the diet did not
affect swine dysentery disease development.
Regardless of fiber effect on post-weaning colibacilosis and swine disentery, the
TRIAL II
main disadvantage of feeding diets with a high content of dietary fiber to pigs is that
these materials tend to affect growth performance negatively. However, negative
effects depend so much on the age of the animals, type of diet and level of inclusion
(Moore et al., 1988; Valencia and Chavez, 1997).
TRIAL III
2.3.1.2. Fermented liquid feed, an example of feed strategy
An interesting strategy to improve pig gut health by dietary manipulation is the
administration of fermented liquid feed, obtained by mixing dry feed with water and
usually, adding bacteria inoculums that act as fermentation starter (Jensen and
Mikkelsen, 1999).
TRIAL IV
It has been demonstrated that administration of fermented liquid feed improves
the performance and gut health of pigs, especially weaner piglets (Geary, 1996;
Brooks et al., 1996; Scholten et al., 1999). Different hypotheses have been proposed
to explain these results. All these hypotheses end from the main characteristics that
TRIAL V
37
INTRODUCTION
Literature review
this feed have: its low pH and high concentration of lactic acid, and its high numbers
The low pH and high amount of lactic acid in the fermented feed is related to a
lower pH of luminal contents of the upper gastrointestinal tract (Ravindran and
Kornegay, 1993) and to higher levels of organic acids (van Winsen et al., 2001). This
LITERATURE
REVIEW
of lactobacilli and yeasts (Adams, 2001).
In particular, some members of the Enterobacteriaceae family that are specially
inhibited by acidic conditions are affected (Jensen and Mikkelsen, 1999). There is a
reduction of enterobacteria in upper gastrointestinal tract that is also maintained in the
lower intestine (vanWinsen et al., 2001), probably due to influences of increased
OBJECTIVES
modification in the gut environment may also influence gut microbiota.
populations of lactobacilli (Urlings, et al., 1993; Van Winsen et al., 2001) and to an
Administration of fermented liquid feed has also been related to a lower total
bacteria population in the stomach and small intestine, and to higher lactic acid
TRIAL I
improvement of colonization resistance mechanisms (Mulder et al., 1997).
bacteria (Jensen and Mikkelsen, 1999). A recent study by Moran and co-workers
(2000) demonstrated a change in the ratio of lactobacilli:coliform throgought
dry-fed animals. Jensen and co-workers also found a lower concentration of coliform
bacteria in the gastrointestinal tract of slaughter pigs fed fermented liquid feed
compared with pigs fed dry feed (1998), and changes in microbiota structure in the
TRIAL II
gastrointestinal tract of piglets, with a significant reduction of coliform compared to
colon of pigs fed fermented liquid feed have also been shown (Leser et al., 2000). In
addition to these results, feeding fermented liquid feed would also affect Brachyspira
administered to pigs (Lindecrona et al., 2003).
In addition to these effects, it is necessary to take into account that when a lactic
TRIAL III
hyodisentariae, showing a lower incidence and severity of the disease when
acid bacteria is used as starter inoculum to produce feed acidification, live and
therefore possibly probiotic lactobacilli are continually fed to the animals, increasing
38
TRIAL V
TRIAL IV
the potential benefits of fermented liquid feed. (Moran et al., 2000; Hillman, 2001).
INTRODUCTION
Chapter 2
2.3.2. Micro-ingredients and in feed additives
LITERATURE
REVIEW
2.3.2.1. Prebiotics
Prebiotics are defined as “non-digestible food ingredients that beneficially affects
the host by selectively stimulating the growth and/or activity of one or a limited
number of bacteria in the colon” (Gibson and Roberfroid, 1995).
To be classified as a prebiotic, a food ingredient must be: 1) neither hydrolyzed
OBJECTIVES
nor absorbed in the upper part of the gastrointestinal tract; 2) a selective substrate for
one or a limited number of potentially beneficial commensal bacteria in the colon,
thus stimulating the bacteria to grow or become metabolically activated, or both; and
3) able as a consequence to alter the colonic microflora toward a more healthier
composition (Collins and Gibson, 1999).
Agarooligosaccharides, fructooligosaccharides, galactooligosaccharides, mannan-
TRIAL I
oligosacharides, xylooligosaccharides, arabinoxylans, raffinose, stachyose, glucosylsucrose, isomalturose, inulin, isomaltose, lactosucrose, lactulose, and lactose are the
main prebiotics used (Patterson and Burkholder, 2003).
Similarly to dietary fiber, prebiotics act by stimulating bacteria fermentation in
the lower gastrointestinal tract, but their mode of action is clearer by thorough
TRIAL II
selective enrichment of specific bacterial populations. According to this definition,
mannan-oligosaccharides are not strictly prebiotics as their mode of action seems to
be due to the neutralization of binding pathogens to mucus receptors and not acting as
specific substrate, however, they have been usually included in this group (Spring et
al., 2000).
TRIAL III
Results using prebiotics have been promising. Studies in vitro have demonstrated
the selective enhancement of the growth of different intestinal bacteria with the
supply of oligosaccharides. Fructooligosacharides increased the growth of
Lactobacillus spp. and Bifidobacterium spp. (Jaskari et al., 1998; Sghir et al., 1998).
Beta-glucooligomers and xylooligomers also improved Lactobacillus spp. and
Bifidobacterium spp. strains (Jaskari et al., 1998), whereas pathogenic and
TRIAL IV
putrefactive bacteria have reduced abilities to degrade these nutrients (Gibson and
Roberfroid, 1995).
In vivo studies have also shown microbial shifts. Farnworth and co-workers
(1992) found numerical increases in total anaerobes, total aerobes, bifidobacteria and
TRIAL V
39
INTRODUCTION
Literature review
coliforms when weanling pigs were fed with inulin. Houdijk et al. (1997) found
studies have also shown improvements in pig growth performance after
administration of different prebiotics (Morimoto et al., 1984; Orban et al., 1997;
Davis et al., 1999, 2004a; Shim et al., 2005).
LITERATURE
REVIEW
decreases in total aerobes in the ileum in response to feeding oligofructose, and other
Modification of the gastrointestinal ecosystem has also been confirmed by
oligosaccharides, oligofructose and transgalactooligosaccharides in the diet of pigs
increased short chain fatty acids production and diminished luminal pH (Bolduan et
al., 1993; Molis et al., 1996; Houdijk et al., 1997; Mikkelsen et al., 2003). The
OBJECTIVES
changes in gut chemical environment. Different studies with non-digestible
selective increase of some bacteria groups also promotes production of their
metabolism, inhibiting the growth of many other species of bacteria (Russell and
These specifically targeted microbial changes can have different beneficial effects
that could explain the growth promoting effect of probiotics. Non-digestible
TRIAL I
Diez-Gonzalez, 1998).
oligosaccharides have been shown to increase resistance to invasion by pathogens in
rats (Bovee-Oudenhoven et al., 1997), reduce translocation of pathogens (Berg,
TRIAL II
1992), and diminish the availability of some toxins in rats (Zhang and Ohta, 1993).
2.3.2.2. Probiotics (live microbial feed supplements)
Probiotics are defined as living micro-organisms in feed which, when taken at
positive effect on the host (Metchinkoff, 1908).
Currently, there are 13 preparations of micro-organisms that are authorized in the
EU as livestock feed additives. Basically, three different groups are used: lactic acid
TRIAL III
certain levels, provide stability of the intestinal flora, and consequently have a
bacteria, mainly Enterococci (Enterococcus faecium), lactobacilli (Lactobacillus
belonging to the genus Bacillus (Bacillus cereus, Bacillus licheniformis and Bacillus
subtilis);
and
Saccharomyces
yeasts
(Sacharomyces
8
9
cerevisiae).
Probiotic
preparations are applied at concentrations of 10 to 10 CFU /kg of feed, mainly in the
TRIAL IV
farciminis and Lactobacillus rhamnosus) and Pediococcus acidilactici; bacteria
40
TRIAL V
form of pelleted mixed feed (Simon et al, 2003).
INTRODUCTION
Chapter 2
Recent research has demonstrated positive effects of probiotics for pigs. Different
studies have shown positive effects on growth performance using different strains
LITERATURE
REVIEW
(Bifidobacterium pseudolongum, Lactobacillus casei, Lactobacillus acidophilus,
Enterococcus faecium, Streptococcus thermophillus, Bacillus spp. and Sacharomyces
spp.; Danek et al., 1991; Kirckgessner et al., 1993; Abe et al., 1995; Kumprecht and
Zobac, 1998; Mathew et al., 1998; Zani et al., 1998; Alexopoulus et al., 2004a,
2004b; Taras et al., 2005), that was accompanied in some cases with reductions in
OBJECTIVES
coliform bacteria and clostridia and with increases in lactobacilli numbers in the gut
(Tortuero et al., 1995; Nemcova et al., 1999). Similarly to these results, Gedek and
co-workers (1993) demonstrated that after the administration of B. cereus to young
pigs, the populations of lactobacilli, bifidobacteria, eubacteria and Escherichia coli in
the upper gastrointestinal diminish, whereas an increase was detected in ileum,
caecum and colon. Despite these promising results with probiotics, it is fair to remark
TRIAL I
that there are also several works that have not found positive effects on pig
performance (Kowarz et al., 1994; Brown et al., 1997; Gardiner et al., 1999).
The usefulness of probiotics in preventing post-weaning diarrhea is also
ambiguous. Whilst some authors have demonstrated a diminution in the incidence of
diarrhea (Zani et al., 1998; Durst et al., 1998; Kyriakis et al., 1999) others have not
TRIAL II
seen such an effect (Eidelsburger et al., 1992; Kirchgessner et al., 1993).
Controversial results may be partly due to the complex etiologic factors involved in
the post-weaning syndrome.
In general, beneficial properties of probiotics have been related to an
improvement of the intestinal microbial balance of pigs and to the strength of the
TRIAL III
indigenous microbiota (Havenaar and Huis In’t Veld, 1992). However, today the
exact mode of action of probiotics is not entirely clear, and different hypothesis have
been postulated.
Probiotics might modulate the intestinal ecosystem by competition with
pathogens for epithelial receptors (competitive exclusion), by competition for
TRIAL IV
nutrients, or by the production of antimicrobial compounds such as bacteriocines and
organic acids with inhibitory effect for undesirable bacteria. Some probiotic effects
have also been related to intestinal immune response stimulation, and also to a
passive aggregation to pathogenic bacteria (Doyle, 2001; Adams, 2001; Simon et al.,
2003).
TRIAL V
41
INTRODUCTION
Literature review
It has also been suggested that probiotics affect the permeability of the gut and
LITERATURE
REVIEW
increase uptake of nutrients (Stewart et al., 1993; Starvic et al., 1995; Lee et al.,
1999).
2.3.2.3. Symbiotics
probiotics and prebiotics in combination (Gibson and Roberfroid, 1995). The live
bacteria must be used with specific substrates for growth. Therefore, the colonization
by an exogenous probiotic could be enhanced and extended by simultaneous
administration of a prebiotic being specifically used by the probiotic strain as a
OBJECTIVES
Another way to modify pig microflora is the use of symbiotics, which is the use of
substrate in the intestinal tract (Rolfe, 2000).
Recently, the administration to weanling pigs of Lactobacillus paracasei in addition
to oligofructose resulted in higher numbers of total anerobes, total aerobes and
TRIAL I
Although works with symbiotics in pigs are still scarce, results are promising.
lactobacilli, with a decrease in enterobacteria and clostridia (Nemcova et al., 1999).
Estrada and co-workers (2001) feeding early-weaned pigs with fructooligosaccharides
resistant starch is not considered as a prebiotic, an interesting result was found by
Brown and co-workers (1997) who demonstrated that concurrent feeding of highamylose corn starch and bifidobacteria to pigs resulted in a higher fecal excretion of
TRIAL II
and Bifidobacterium longum found an improvement in feed efficiency. Although
bifidobacteria than when the probiotic was administered alone. In addition to the
increase of substrate for bacteria, it seems that the effect found, might be due to the
carrier through the gastrointestinal tract (Crittenden, 1999).
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bifidobacteria attachment to the surface of the starch granules that might be act as a
2.3.2.4. Acidifiers
propionic, butyric, lactic, fumaric, Ca-formate, Ca-propionate, K-diformate, and Nabenzoate (Mroz, 2003).
42
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modulators of the gut ecology in pigs. Some of the most used are: formic, acetic,
TRIAL IV
Several organic acids and their salts are recognized as preservatives and
INTRODUCTION
Chapter 2
Organic acids and their salts appear to be potential alternatives to prophylactic infeed antibiotics for improving the performance of weaned piglets, fattening pigs and
LITERATURE
REVIEW
reproductive sows. As with other feed additives however, acidifiers are mainly used
in young pigs as a way to prevent the problems associated with early weaning
(Maxwell and Carter, 2001). In this regard, the administration of organic acids has
also been reported to be helpful in overcoming problems of the post-weaning period
in piglets (Partanen and Mroz, 1999; Tsiloyiannis et al., 2001).
OBJECTIVES
Moreover, administration of organic acids, such as formic, acetic, propionic,
lactic, citric, fumaric, sorbic, tartaric and malic acid, and some of their salts on
growth performance are well defined (Han et al., 1998; Radcliffe et al., 1998;
Siljander-Rasi et al., 1998; Øverland et al., 1999; Bosi et al., 1999).
The exact mechanism of action of organic acids remains unclear, although several
hypotheses have been postulated. The primary antimicrobial action of organic acids
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(strain-selective growth inhibition or delay) is through pH depression of the diet,
acting as a preservative, inhibiting the growth of many species of bacteria, yeasts and
moulds on the feed previously its consumption. The specificity depends on the type of
acid used; whilst acetic acid has demonstrated a broad spectrum and inhibits growth
of bacteria, yeast and moulds, the action of propionic acid is primarily against
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moulds, with poor activity against bacteria and none against yeasts (Foegeding and
Busta, 1991, Partanen, 2001).
However, more important than preservative action of organic acids, is their action
by different direct effects on the animal. One of the first mechanisms proposed was
the acidification of the digesta, particularly in the stomach of young pigs which have
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a limited secretion of HCl in the first stages of life (Maxwell and Carter, 2001).
Nevertheless, the evidence suggests that this gut pH reduction is not the main effect
of these compounds (Risley et al., 1992, Roth et al., 1992; Partanen and Mroz, 1999)
and several trials have failed to demonstrate reductions of digesta pH after the
inclusion of different acids in the diet (Risley et al., 1991; Gubert and Sauer, 1995;
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Franco et al., 2005).
The ability of acids to change from undissociated to dissociated form, depending
on the environmental pH,
has been recently proposed as the most plausible
mechanism of action. This capacity makes organic acid effective antimicrobial agent
in the pig gut. Undissociated form become lipophilic and can freely diffuse through
TRIAL V
43
INTRODUCTION
Literature review
the bacteria membrane into their cytoplasm (Partanen, 2001). Once inside the cell, the
enzymes responsible of nutrient uptake (Cherrington et al., 1991; Russell, 1992).
Organic acids with higher pKa values are more effective and their antimicrobial
efficacy is generally improved as chain length and degree of unsaturation increase
LITERATURE
REVIEW
acid dissociates and suppresses different bacterial enzymes such as ATPases and
(Foegeding and Busta, 1991). Overall, the antimicrobial activity is primarily against
acid bacteria are more resistant to their effects (Lueck, 1980).
Different in vivo studies have demonstrated microbiota shifts when acidifiers are
used. Specifically, different types of organic acids are related to marked reductions in
OBJECTIVES
yeasts and bacteria belonging to the Enterobacteriaceae family (Frank, 1994). Lactic
pig coliform bacteria (Mathew et al., 1996; Jensen, 1998; Øverland et al., 1999;
Øverland et al., 2000). In the upper and lower intestine, micro-organism counts of
et al., 1992). However, results regarding microbial shifts are not consistent and some
authors have not found changes when administering different acidifiers to pigs
TRIAL I
lactobacilli, bifidobacteria, and eubacteria have also been shown to decrease (Gedek
(Bolduan et al., 1988; Risley et al., 1992).
In addition, organic acids may be used as energetic substrate or as modulator for
also as precursors for synthesis on non-essential amino acids, DNA and on lipids
required for intestinal growth (Mroz, 2003).
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mucosal development, epithelial cell growth and increasing absorptive capacity, and
Dietary supplementation with high levels of minerals such as copper and zinc has
usually been used in piglets to modulate intestinal microbiota and improve
gastrointestinal health. Administration of pharmacological doses of ZnO have been
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2.3.2.5. Minerals: zinc and copper
related to improvements in post-weaning performance (Hahn and Baker, 1993; Hill et
al., 1996; Smith et al., 1997; Mahan et al., 2000; Hill et al., 2000; Case and Carlson,
Waern et al., 1998).
The effects shown may be due to microbiota modulation. The supplementation of
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2002) and preventing the apparition of post-weaning diarrhea (Poulsen, 1998; Jensen
high doses (2500 ppm) of zinc oxide to piglets has been related to increases in the
44
TRIAL V
biodiversity of coliforms and increases in the stability of pig microbiota (Katouli et
INTRODUCTION
Chapter 2
al., 1999). Recently, the same doses have shown a reduction in the total number of
anerobes and in lactic acid bacteria in the stomach and ileum, parallel with an
LITERATURE
REVIEW
increase in coliform and enterococci throughout the gastrointestinal tract (Höjberg et
al., 2005).
Similarly to ZnO, administration of pharmacological doses of copper sulphate
has shown improvements in feed efficiency and weight (Cromwell et al., 1989; Dove
and Hayden, 1991; Dove, 1995; Hill et al., 2000). Again, the improvement in growth
OBJECTIVES
performance was related to an antimicrobial action of copper (Fuller et al., 1960). A
reduction in lactic acid bacteria and lactobacilli throughout the gastrointestinal tract
and in colonic coliform bacteria (Höjberg et al., 2005) have been shown recently.
However, it is possible that beside the potential antimicrobial effects, benefits of
copper sulphate and also zinc oxide are due to a systemic effect; Zhou and co-workers
(1994a) observed an increase in gain when pigs were injected intravenously with
TRIAL I
copper.
2.3.2.6. Plant extracts
For thousands of years, herbs and spices containing essential oils have provided
TRIAL II
distinctive properties to foods, and many have proved to be potent antimicrobial
agents. Some of the most common plant products known for their antimicrobial
properties belong to the genus Allium, and include garlic, onion and leek; others are
thyme, oregano, marjoram, basil, cumin and bay. There are also spices, of which
cloves, cinnamon, pepper and nutmeg may be remarked.
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In vitro studies have demonstrated antibacterial activity of different plant extracts
(Sen et al., 1998; Dorman and Deans, 2000; Friedman et al., 2002), and in recent
years, the inclusion of these products in the pig diet has been proposed as a means to
prevent intestinal disorders, especially at weaning, and to promote growth.
However, effects on growth response are not consistent and depend so much on
the plant used. Different studies have added garlic to weanling pig diets without
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positive influence on performance (Horton et al., 1991; Holden et al., 1998; Holden
and McKean, 2000), although a promising effect reducing post-weaning mortality has
been found (Peet-Schwering et al., 2000). Similarly, a positive effect on growth
performance of weanling pigs was also shown by Cromwell and co-workers (1985),
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45
INTRODUCTION
Literature review
after administration of yucca plant extract, and herbal mixtures (great nettle, garlic
Effects on performance are generally attributed to an effect of plant extracts on
intestinal microbiota. In this regard, Tedesco and co-workers (2005) recently found
an improvement in weaning pigs performance and also marked changes in microbiota
LITERATURE
REVIEW
and wheat grass; Grela et al., 1998).
composition (lower total bacteria, E. coli, total anerobic and Enterococcus spp. in
Taeonia lactiflora, Olea europea and Portulaca oleracea.
2.3.2.7. Other additives
OBJECTIVES
feces) when different herbal additives were added to the feed (Lycium barbarum,
There are several other additives that are being used in pig feed, of which
as a result of the inclusion of enzymes in animal diet (Bedford and Schulze, 1998).
The main effect of using enzymes is a high availability of dietary nutrients for the
TRIAL I
enzymes are among the most important. Many beneficial effects have been reported
animal (Verstegen and Williams, 2002), which help digestion and the breaking down
of substrates that could provoke excessive microbial fermentation and disturb
regarding microbial shifts after enzymes addition in pigs, evidence in poultry suggests
that an effect may be expected. The addition of xylanase to broiler diets reduced total
ileal bacteria numbers by 60% and also reduced the proportion of bacteria with lower
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microbial equilibrium (McCartney, 2005). Although there is no known evidence
Summary
Concerns regarding resistance in human bacterial pathogens have led to the total
TRIAL III
guanidine:cytosine content (Apajalahti et al., 2001).
ban of antibiotics as growth promoters in animal feed on January 2006. As a
consequence important efforts have been made to look for alternatives or replacement
the maintenance of the gastrointestinal ecosystem in pigs. The use of different
strategies has been proposed with positive results, among them may be remarked
different sources of fiber, prebiotics, probiotics, organic acids, mineral at
TRIAL IV
strategies to improve growth performance and to control enteric bacterial diseases by
46
TRIAL V
pharmacological doses and plant extract mixtures. A better understanding of the
INTRODUCTION
Chapter 2
modes of action of these products will allow in the near future a more rational design
of non-antibiotic growth promoters.
LITERATURE
REVIEW
OBJECTIVES
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INTRODUCTION
Literature review
2.4.2.4. New tools for the analysis of the gastrointestinal microbiota
Introduction
LITERATURE
REVIEW
1.4.
Traditionally, gut microbiota has been studied by classical selective-culture
classical methods to detect all gut community bacteria has been established by
different studies: low sensitivities, inability to detect non-cultivatable bacteria and
unknowns species, time-consuming aspects and low levels of reproducibility
OBJECTIVES
methods based on phenotytic characterisation. However, the inadequacy of these
(Fuhrman et al., 1992; Dutta et al., 2001). Recent studies have demonstrated that
only 10-40% of total gut bacteria are culturable (Zoetendal et al., 1998; Suau et al.,
bacteria isolates is often inadequate (Leser et al., 2002).
In order to overcome the problems mentioned above, higher resolution molecular
TRIAL I
1999) and that the classical taxonomy based on physiological/biochemical analysis of
techniques based on 16S ribosomal DNA genes have been developed in recent years
(Amann et al., 1995). The introduction of these methods in gastrointestinal
such as the pig gut microbiota (Vaughan et al., 2000). Nowadays, the 16S rRNA gene
is the key bacterial marker due to its genetic stability, its domain characteristic
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microbiology has greatly enhanced our knowledge of complex microbial populations
composition with highly conserved and variable regions, and its high copy number in
bacterial cell (Woese, 1987; Amann et al., 1990a). In addition, the growth of the
sequence
data
bank
(www.ncbi.nlm.nih.gov/entrez/,
rdp.cme.msu.edu/index.jsp) allows easy comparison between sequences from across
the world. The use of these new methods has been especially focused on the
description of the different species inhabiting the gastrointestinal tract and also on the
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genes
detection of bacterial shifts related to parameters such as age, diet or illness.
works involving molecular methods for studying pig gut microbiota.
48
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methods habitually applied in gut microbiology, paying special attention to recent
TRIAL IV
This chapter will focus on different quantitative and qualitative molecular
INTRODUCTION
Chapter 2
2.4.1. Quantitative techniques
LITERATURE
REVIEW
Several different techniques may be used to quantify bacteria in the
gastrointestinal tract. These methods can be classified basically into two groups:
quantitative PCR-based methods such as real time PCR, and other methods not
dependent on this previous amplification but on the use of labelled probes, such as
fluorescent in situ hybridization, dot blot and microarrays. Of these, we will look
closely at two methods in this chapter due to their relatively more frequent use in
OBJECTIVES
gastrointestinal microbiology in the last few years: real time PCR and fluorencent in
situ hybridization.
2.4.1.1. Quantitative Polymerase Chain Reaction (qPCR)
Real-time PCR is a method based on the polymerase chain reaction with on-line
TRIAL I
measurement of the amplification reaction. Data are automatically collected
throughout the entire PCR process, rather than at the end of the PCR as conventional
reaction was traditionally performed.
Real-time PCR system is based on the detection and quantification of a
fluorescent reporter (Lee et al., 1993; Livak et al., , 1995) that increases its signal in
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direct proportion to the amount of PCR product in the reaction. Quantification of the
product takes place in the exponential phase of PCR, where the first significant
increase in the amount of PCR product correlates to the initial amount of target
template. The higher the starting copy number of target DNA, the sooner a significant
increase in fluorescence is detected. Absolute quantitation can be achieved by
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interpolating unknown samples from a standard curve constructed with a known
amount of the target gene.
There are two different methods of real-time PCR (Figure 2.5). The one that first
appeared was the TaqMan assay (Holland et al., 1991). This method is based on the
use of a fluorogenic labelled probe in addition to both primers that are essential in the
PCR reaction. TaqMan probes are oligonucleotides that contain a fluorescent dye
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usually on the 5’end, and a quenching dye (usually TAMRA) on the 3’ end, and it is
designed to anneal to an internal region of the PCR product. When this probe is
irradiated, the excited fluorescent dye transfers energy to the quenching dye molecule
rather than fluorescing (Hiyoshi and Hosoi, 1994). When the target sequence is
TRIAL V
49
INTRODUCTION
Literature review
present, the probe anneals downstream from one of the primer sites and is cleaved by
cleavage produces a separation from the two dyes that mark the probe and the
reporter dye starts to emit a signal that increases in each cycle proportional to the rate
of probe cleavage. At the same time, Taq polymerase removes the probe from the
LITERATURE
REVIEW
the 5’ nuclease activity of the Taq polymerase as the primer is extended. This probe
target, allowing extension to end the template strand. With each PCR cycle,
an increase in fluorescence intensity proportional to the amount of amplicon
produced.
The second real-time PCR system is the SYBR Green dye. This method uses a
OBJECTIVES
additional reporter dye molecules are cleaved from their respective probes resulting in
non-sequence specific fluorescent intercalating agent (SYBR Green) that only emits
when bound to double stranded DNA. SYBR Green dye is a fluorogenic minor
signal upon binding to double-stranded DNA (Morrison et al., 1998). During the
PCR, the polymerase amplifies the target sequence, creating new PCR products.
TRIAL I
groove binding dye that shows little fluorescence when in solution, but emits a strong
Then, the SYBR Green dye binds to each new copy of double-stranded DNA. As the
PCR progresses, more amplicons are created, increasing the intensity of fluorescence
The main difference between both methods is that SYBR Green chemistry detects
all double-stranded DNA, including non-specific reaction products, and primer-
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detected with each new amplification cycle.
dimer, making it therefore more important to have a well-optimized reaction so as not
to obtain unspecific amplification that may generate false positive signals. This
although it can be minimised since non-specific amplification can be easily discarded
by analysis of melting or dissociation curve of the product amplified (Ririe, 1997).
On the other hand, the main disadvantage of TaqMan chemistry is that the synthesis
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problem is especially important with low quantity template (Hein et al., 2001),
of different probes is required for each different sequence that wants to be detected.
Real-time PCR methodology has different advantages in bacteria quantification
compared to traditional culture: higher sensitivity, rapidity and reproducibility (Bustin
et al., 2000). The possibility of storing the samples until their analysis, avoiding the
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This increases the assay set-up and running costs.
need to work in fresh, is undoubtedly a remarkable advantage of this method
50
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compared to traditional ones. However, this method tends to overestimate bacterial
INTRODUCTION
Chapter 2
populations ((Nadkarni et al., 2002; Huijsdens et al.,2002) and also has a relatively
high cost.
LITERATURE
REVIEW
Figure 2.5. Representation of real-time PCR with TaqMan primers (A) and SYBR
Green (B). (A). In the intact TaqMan probe, energy is transferred from the shortwavelength fluorophore (green circle) to the long-wavelength fluorophore (red
circle), quenching the short-wavelength fluorescence. After hybridization, the probe
is susceptible to degradation by the endonuclease activity of a Taq polymerase. Upon
OBJECTIVES
degradation, quenching is interrupted, modifying the fluorescence detected. (B).
SYBR Green I dye (black diamonds), present in the PCR mixture, becomes
fluorescent (green diamonds) upon binding to all double-stranded DNA, providing a
direct method for quantifying PCR products in real time (Invitrogen PCR Handbook).
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A
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B
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51
INTRODUCTION
Literature review
In recent years, real-time PCR has been widely used to quantify selective bacteria
Ott et al., 2004; Penders et al., 2005), pigs (Collier et al., 2003; Hill et al., 2005),
chickens (Selim et al., 2005; Wise and Siragusa, 2005) and ruminants (Tajima et al.,
2001), and also to detect pathogen bacteria in different environments such as water,
LITERATURE
REVIEW
from the gastrointestinal tract of humans (Huijsdens et al., 2002; Matsuki et al., 2003;
feces and soil (Smythe et al., 2002; Ibvekwe and Grieve, 2003; Fukushima et al.,
Particularly in pigs, real time PCR is being used to quantify total bacteria and
some specific bacterial groups. Collier and co-workers (2003) used real-time PCR
with SYBR Green dye chemistry to quantify total bacteria and Lactobacillus spp. in
OBJECTIVES
2003; Wu et al., 2005).
the ileal and colonic contents of growing barrows fed with different experimental
diets. Real time PCR allows the detection of significant differences in total bacteria
workers (2005) quantified different pig gut bacteria belonging to the Bacillales,
Clostridium spp., Streptococcus alactolyticus, and Lactobacillus amylovorus using
TRIAL I
and lactobacilli bacteria in pigs fed the experimental diet. More recently, Hill and co-
real time PCR and also SYBR Green chemistry in weanling pigs fed different diets.
In this case, Chaperonin-60 gene was used as a targeted gene instead of the 16S
changes in specific pig gut bacteria and the potential of Chaperonin-60 gene as an
alternative to the use of 16S rDNA gene in microbial ecology.
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rDNA gene. This study demonstrated the usefulness of real time PCR for detecting
2.4.1.2. Fluorescent In Situ Hybridization (FISH)
an increasing interest in gut microbiology. It was first used in bacteriology by
Giovannoni and co-workers (1988) with radioactively labelled oligonuclotide probes.
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Fluorescent In Situ Hybridization (FISH) is a quantitative molecular method with
Although the basis of the method has not changed, fluorescent probes have now
replaced radioactive ones. FISH method detects nucleic acid sequences by a
sequence within the intact bacterial cell (Moter and Göbel, 2000). Depending on the
specificity of the probe used, different specific bacterial groups can be counted; if
universal probes are used, total bacteria can be quantified. To date, several probes
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fluorescently labelled probe that hybridizes specifically to its complementary target
have been standarized and are actually being used to quantify the main gut bacteria
52
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(Table 2.5).
INTRODUCTION
Chapter 2
FISH procedure is relatively easy though laborious. Bacterial cell is chemically
treated to allow cell fixation and permeabilization. Once fixed, bacterial cells are
LITERATURE
REVIEW
immobilized on a pre-treated glass slide or kept in suspension depending on the
method of quantification that will be used afterwards. Then, hybridization under
stringent conditions allows proper annealing of the selected probe to the target
sequence. Generally, probes are 15-30 nucleotides in length and covalently labelled at
the 5’ end with a fluorescent dye. Common fluorophors include fluorescein,
OBJECTIVES
etramethylrhodamine, Texas red, and carbocyanine dyes such as Cy3 and Cy5
(Southwick et al., 1990). Nowadays, two different methods are used to quantify
stained cells. The most common method used is via epifluorescence microscopy
though it is laborious and subjective (Wagner et al., 2003). Alternatively, flux
cytometry appears as a potential method with high-resolution to bacteria counts.
One of the main advantages of FISH is that being a molecular method it does not
TRIAL I
depend on purification or amplification steps, avoiding biases that are typically
described on PCR based methods (Wintzingerode et al., 1997).
Another advantage, when microscope slides are used, is that counts can be made
using a confocal laser scanning microscope, obtaining an accurate image of the
spatial distribution of microbial communities as well as information about
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morphology (Moter and Göbel, 2000; Daims et al., 2001). Moreover, when the
technique is standarized, counting can be automatized, thus avoiding the biases that
manual counts can produce (Jansen et al., 1999). In addition, recently a multi-color
fluorescence in situ hybridization method has been developed, which detects, in a
single reaction, seven species of Bifidobacterium (B. adolescentes, B. angulatum, B.
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bifidum, B. breve, B. catenulatum, B. dentium, and B. longum; Takada et al., 2004,
Figure 2.6). This approach may be an interesting alternative to quantify different
groups of bacteria at a time in digesta samples.
Especially interesting is the recent application in gastrointestinal microbiology of
flow citometry to FISH signal detection, which allows a relatively faster and more
TRIAL IV
sensitive quantification than traditional microscopy (Wallner et al., 1997).
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53
Table 2.5. Some probes currently used to quantify different gastrointestinal bacteria.
Probe name
Target group
Probe sequence( 5’-3’)
Reference
S-D-Bact-0338-a-A-18
S-*-Bacto-0303-a-a17
S-S F.suc-0650-a-A-20
S-S-F.int-0136-a-A-20
S-*-F.prau 0645-a-A-23
S-*-Erec-0482-a-A-19
S-*-Elgc-01-a-A-19
S-*-Chis-0150-a-A-23
S-*-Bdis-0656-a-A-18
S-*-Bfra-0602-a-A-19
S-S-Bvulg1017-a-A-21
S-*-Bacto-1080-a-A-18
S-G-Bif-0164-a-A-18
S-*-Rfla-729-a-A-18
S-*-Rbro-730-a-A-18
S-*-Ehal-1469-a-A-18
Bacteria
CFB phylum
F. succinogenes
F. intestinalis
F. prausnitzii
C. coccoides cluster
C. leptum cluster
C. histolyticum
Bacteroides distansonis
Bacteroides fragilis
Bacteroides vulgatus
Bacteroides spp.
Bifidobacterium spp.
Ruminococcus albus and R. flavefaciens
C. sporosphaeroides, R. bromii, C. leptum
Eubacterium halii group
GCTGCCTCCCGTAGGAGT
CCAATGTGGGGGACCTT
TGCCCCTGAACTATCCCAAGA
CGGTTGTTCCGGAATGCGGG
CCTCTGCACTACTCAAGAAAAC
GCTTCTTAGTCAGGTACCG
GGGACGTTGTTTCTGAGT
TTATGCGGTATTAATCTYCCTTT
CCGCCTGCCTCAAACATA
GAGCCGCAAACTTTCACAA
AGATGCCTTGCGGCTTACGGC
GCACTTTAAGCCGACACCT
CATCCGGCATTACCACCC
AAAGCCCAGTAAGCCGCC
TAAAGCCCAGYAGGCCGC
CCAGTTACCGGCTCCACC
Amann et al., 1990b
Manz et al., 1996
Amann et al., 1990a
Lin et al., 1994
Suau et al., 2001
Franks et al., 1998
Franks et al., 1998
Franks et al., 1998
Franks et al., 1998
Franks et al., 1998
Rigottier-Gois et al., 2003
Doré et al., 1998
Langedink et al., 1995
Harmsen et al., 2002
Harmsen et al., 2002
Harmsen et al., 2002
S-G-Lab0158-a-A-21
Lactobacillus-Enterococcus spp.
GGTATTAGCAYCTGTTTCCA
Harmsen et al., 2002
L-S-E.coli-1531-a-A-21
Escherichia coli
CACCGTAGTGCCTCGTCATCA
Krogfelt et al., 1993
S-*-Enter-1432-a-A-15
Enterobacteriaceae
CTTTTGCAACCCACT
Sghir et al., 2000
a
Probe names have been standarized as follows: S or L for Large or Small subunit rDNA as the target; D for Domain, O for Order, F for Family, G for Genus, S
for Species and Ss for Subspecies. ; letters designating the target group of the oligonucleotide probe; nucleotide position in E. coli gene; letter designating the
version of the probe; S or A for Sense or Antisense direction; number indicating the length in nucleotides of the probe (Alm et al., 1996). An asterisk shows that
this probe has not been standarized.
INTRODUCTION
LITERATURE
REVIEW
INTRODUCTION
Chapter 2
However, the FISH methods have also some limitations. One of them is the
different penetration of probes in bacteria with various cell wall types. The high
LITERATURE
REVIEW
complexity of gram-postitive bacteria wall hinders its permeabilization and probe
hybridization which can result in an underestimation of these bacteria (Langendijk et
al., 1995; Jansen et al., 1999).
Figure 2.6. (A) Epifluorescent image of mixed culture of seven different
OBJECTIVES
Bifidobacterium species by multi color FISH. In the images, B. adolescentis, B.
angulatum, B. bifidum, B. breve, B. catenulatum, B. dentium and B. longum are
shown in G (green), R (red), C (cyan), B (blue), Y (yellow), M (magenta) and W
(blue-ish white), respectively. (B) Identification of Bifidobacterium species in human
fecal samples using multi color FISH (Takada et al., 2004).
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A
B
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FISH methods have been used in different microbiological studies. FISH has been
used extensively to identify and count bacteria, specially from marine environments
(Ramsing et al., 1996; Glöckner et al., 1996; Alfreider et al., 1996; Lemke et al.,
1997; Jürgens et al., 1999), and also to study microbial diversity in wastewater
treatment (Amann et al., 1996; Snaidr et al., 1997; Bond et al., 1999). Moreover FISH
TRIAL IV
has been used in the study of different complex bacterial ecosystems in the human
body such as those of the oral cavity (Moter et al., 1998a, 1998b) and the
gastrointestinal tract (Langedijk et al., 1995; Franks et al., 1998; Harmsen et al.,
1999; Jansen et al., 1999; Harmsen et al., 2000a, 2000b, 2002; Zoetendal et al.,
2002a; Hold et al., 2003). Finally, FISH has been also applied in the detection of
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INTRODUCTION
Literature review
pathogens in tissue samples (Boye et al., 1998; Trebesius et al., 1998; Jensen et al.,
Although FISH has been extensively used to study human intestinal microbiota,
only a few published works can be found on the study of pig gut bacteria.
Konstantinov and co-workers (2004b) used a universal probe to quantify total
LITERATURE
REVIEW
2000) and in feces (Waar et al., 2005).
bacteria (Bac338), and a probe to quantify Lactobacillus-enterococcus group
to the genera Lactobacillus amylovorus and L. reuteri-like in weanling pigs that were
receiving diets rich in fermentable carbohydrates. Utilization of FISH allowed
determination of a higher prevalence of L. reuteri and L. amylovorus-like populations
OBJECTIVES
(Lab158) and developed and evaluated a specific probe to quantify bacteria belonging
2.4.2. Fingerprinting techniques: DGGE, t-RFLP
Fingerprinting techniques are based on the existence of polymorphisms in the 16S
TRIAL I
in the ileum and colon of pigs fed diets rich in fermentable carbohydrates.
rDNA gene within different bacteria, which provide specific patterns or profiles for
each microbial community depending on the bacteria harbouring it. Genetic
dynamics and diversity of complex bacterial populations. Terminal restriction
fragment length polymorfism (T-RFLP) and denaturing gradient gel electrophoresis
(DGGE) are one of the most used.
TRIAL II
fingerprinting techniques are actually being used to elucidate the complexity,
T-RFLP is based on comparison of banding patterns obtained from DNA
(Charteris et al., 1997; Hozapfel et al., 2001), and DGGE is based on the separation of
an amplified fragment of the 16S rDNA in a denaturing electrophoresis depending on
its sequence (Muyzer et al., 1993).
TRIAL III
restriction with an endonuclease that recognize specific sequences within the gene
Denaturant/Temperature Gradient Gel Electrophoresis allows separation of
objective DNA molecules based on variability of its sequence in the variable regions
TRIAL IV
2.4.2.1. Denaturant/Temperature Gradient Gel Electrophoresis (DGGE/TGGE)
and thus in its chemical stability of 16S rDNA. First introduced in microbial ecology
56
TRIAL V
by Muyzer and co-workers (1993) it is widely used currently.
INTRODUCTION
Chapter 2
In these techniques, PCR-amplified 16S rDNA products are separated by applying
a temperature gradient or denaturing gradient in an electrophoresis system. A
LITERATURE
REVIEW
temperature or chemical gradient is established in a polyacrylamide gel in parallel to
the electric field. The DNA samples migrate through the gradient from low to high
temperature, or low to high chemical gradient. At the point in the gradient where
partial denaturation of the double-stranded DNA happens, the migration of the DNA
fragment is drastically retarded and sequences of the same size, but of different
OBJECTIVES
thermal or chemical stability (by its sequence), are separated (Reisner et al., 1992).
Separation is therefore based on the melting of the DNA fragments. Sequence
variation causes the melting temperatures to differ, and molecules with different
sequences will stop migrating at different positions in the gel (Muyzer and Smalla,
1998).
DNA bands are thereafter visualised using ethidium bromide, silver staining or
TRIAL I
SYBR Green I. The PCR banding pattern is indicative of the number of bacterial
species that are present and thus allows visualization of the genetic diversity of
microbial populations (Simpson et al., 1999). Subsequent identification of specific
bacterial groups or species in the sample can be achieved by cloning and sequencing
the excised bands from the gel, or by hybridization of the profile using phylogenetic
TRIAL II
probes (Muyzer and Smalla, 1998).
Both methodologies have been successfully used in gut microbial studies due to
the fact that these techniques are reliable, rapid, comparatively inexpensive and with
good reproducibility (Ampe et al., 2001; Schmalenberger et al. 2001; McCartney,
2002). These techniques have been used in human samples (Zoetendal et al., 1998;
TRIAL III
Zoetendal et al., 2002b; Favier et al., 2002; Malinen et al., 2003; Gueimonde et al.,
2004), in pig gastrointestinal samples (Simpson et al., 1999; Simpson et al., 2000;
Collier et al., 2003; Konstantinov et al., 2003; Konstantinov et al., 2004b; Inoue et al.,
2005), in “in vitro” modification of pig microbiota after substrates inoculums (Zhu et
al.., 2003) and to study bacterial biofilms (Muyzer et al., 1993; Muyzer and de Waall,
1994).
TRIAL IV
Different groups have been using DGGE to study pig gut microbiota. The first
work using this technique was done by Simpson and co-workers (1999, 2000; Figure
2.7), with the aim of determine if DGGE could be effectively applied to measure
changes in bacterial populations in the gastrointestinal tract, based upon age, diet, or
TRIAL V
57
INTRODUCTION
Literature review
anatomic compartment. The authors concluded suitability of the method as DGGE
different ages and among individual gut compartments. Difference in patterns
observed have also been elucidated after administration of different fermentable
carbohydrates to weanling pigs (Konstantinov et al., 2003, 2004b), showing an
LITERATURE
REVIEW
analysis revealed diverse and stable individual bacterial populations between pigs of
increase in microbial stability and higher diversity. Similarly, Zhu and co-workers
piglets as inoculum of sugar beet pulp fermentation. Collier and co-workers (2003)
have also been using this technique to determine differences in pig colonic and ileal
microbiota after an antibiotic growth promoter administration. Differences in band
patterns demonstrated the effect of the antibiotic compared to control pigs. Recently,
OBJECTIVES
(2003) found differences in band patterns in an in vitro study with feces of weaning
an interesting study have found changes in piglet microbial profiles during the first
weeks of life using DGGE, detecting remarkable changes in microbiota diversity and
TRIAL I
composition (Inoue et al., 2005).
Figure 2.7. PCR-DGGE profile generated from fecal samples obtained from an
individual piglet over a 20-day experimental period using primers specific for the V3-
58
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TRIAL II
16S rDNA. Bacterial standard marker lanes are denoted as M (Simpson et al., 2000).
INTRODUCTION
Chapter 2
2.4.2.2. Terminal Restriction Fragment Length Polymorfism
LITERATURE
REVIEW
Terminal restriction fragment length polymorphism is a very useful tool for
comparing microbial communities (Kitts, 2001) that allows the fingerprinting of a
community by analyzing the polymorphism of the 16S rDNA. It is a high-throughput,
reproducible method that allows a qualitative analysis of the diversity of bacteria in
an ecosystem.
Firstly, DNA from the sample is extracted and by the use of universal primers,
OBJECTIVES
total bacterial DNA is amplified by conventional PCR, similarly to DGGE with the
difference that one of the primers used is labelled fluorescently at the 5’ end. The
amplified DNA is then digested with a restriction enzyme, which is an endonuclease
that recognizes one determined specific sequence into the amplicon. Once the
restriction is obtained, fragments are separated by capillary electrophoresis.
Generally, a DNA sequencer with a fluorescence detector is used to separate
TRIAL I
fragments, thus, T-RFLP gives only one band per species as only the fragment
containing the fluorescently labelled primer site will be detected (Figure 2.8). The
samples are run on long sequencing gels that give high resolution and sensitive
detection. Once electropherogram is obtained, inference of potential bacteria present
in the sample can be achieved by comparison of fragments obtained in the samples
TRIAL II
with in silico restriction with the primers and enzyme used, using the analysis
function TAP-tRFLP from the Ribosomal Database Project II software (Cole et al.,
2003).
T-RFLP has appeared recently as an attractive tool for studying pig gut
microbiota (Leser et al., 2000; Khan et al., 2001; Hogberg et al., 2004), chicken
TRIAL III
microbiota (Gong et al., 2002), human microbiota (Gong et al., 2003; Nagashima et
al., 2003; Ott et al., 2004; Wang et al., 2004), rats microbiota (Kaplan et al., 2001),
and characterization of bacteria from environmental samples (Liu et al., 1997;
Clement et al., 1998; Dunbar et al., 2000; Blackwood et al., 2003).
Particularly in pigs, different studies have used t-RFLP to study gut microbiota.
TRIAL IV
Leser and co-workers (2000) compared bacterial communities in the colon of pigs fed
different experimental diets based on either modified standard feed or cooked rice
supplemented with dietary fibers. After feeding animals with the experimental diets,
differences in bacterial community structure were detected as different patterns were
obtained. Similarly, Högberg and co-workers (2004) studied the effect of different
TRIAL V
59
INTRODUCTION
Literature review
cereal non-starch polysaccharides on the gut microbiota in growing pigs. The authors
and co-workers (2005) also found differences in microbial cecal profiles in pigs
receiving different doses of zinc oxide and copper sulphate (Figure 2.9).
LITERATURE
REVIEW
observed a particular pattern depending on the diet administered. Recently, Höjberg
obtained after an enzymatic restriction. Bars represent different sequences, red spirals
indicate fluorescent label; and circles, squares and rectangles indicate different
restriction enzyme sites and their location in each sequence. The fragment analysis
As is with other PCR based methods, both these fingerprinting techniques (t-RLP
TRIAL II
TRIAL I
peaks would look like the graph on the right.
OBJECTIVES
Figure 2.8. An example of fragments and visualization of the electropherogram
and DGGE) are highly determined by the pair of primers chosen for PCR
amplification. Primers chosen will limit the number of targeted DNA and thus may
an overall description of an ecosystem wants to be achieved. Specifically with tRFLP, the selection of primers and restriction enzyme is particularly important. An
TRIAL III
bias the profile obtained, being especially important to select universal primers when
inappropiate selection can make that many bacteria species share the same length of
fragment, avoiding therefore the precise recognition of all the microbial diversity (Liu
Summary
TRIAL IV
et al., 1997; Marsh, 1999; Kaplan et al., 2001).
In recent years, the increasing concern on gut health has rekindled the interest for
gut bacteria. This fact with the inadequacy of classical culture-dependent methods to
60
TRIAL V
accurately describe all microorganisms, has overcome the development of different
INTRODUCTION
Chapter 2
molecular methods to study gastrointestinal microbiota. As a consequence, nowadays
there is a current plethora of genetic techniques for quantification, identification and
LITERATURE
REVIEW
community characterisation with a huge amount of information regarding the
ecosystem and how it changes with age, illness and dietary modification. Although
the majority of works are still concentrating on human microbiota, important efforts
are being made to apply these methods on pig gut; this is significantly increasing our
microbiota knowledge and in the near future will provide important information
OBJECTIVES
regarding the key role of gut bacteria for animal health.
Figure 2.9. T-RFLP profiles obtained from cecum digesta in pigs receiving
different doses of zinc oxide and copper sulphate (Höjberg et al., 2005).
TRIAL I
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TRIAL II
TRIAL I
62
OBJECTIVES
OBJECTIVES
TRIAL V
LITERATURE
REVIEW
INTRODUCTION
Objectives
Chapter 3
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
2. To study microbiota establishment and changes produced in the piglet by
weaning, and the quantitative importance of major bacterial groups along the
gastrointestinal tract in the growing animal.
3. To study potential modification of this ecosystem by the inclusion of different
commercial in-feed additives or fibrous ingredients in the diet of weaned or growing
pigs.
INTRODUCTION
LITERATURE
REVIEW
1. To develop and/or evaluate molecular methods to study pig gut microbiota:
real time PCR and fluorescence in situ hybridization (FISH) to quantify particular
microbial groups, and terminal-restriction fragment length polymorfism (t-RFLP) to
determine global changes in the community profile.
OBJECTIVES
The main objective of this thesis was to improve our knowledge of pig gut
microbiota, bearing in mind the development of new feeding strategies to substitute
antibiotics as growth promoters. To achieve this, three secondary objectives were
considered:
TRIAL I
Objectives
Trial III was designed with two different objectives. Firstly, to describe the main
bacteria groups throughout the gastrointestinal tract of the growing pig using FISH as
a method of study; and secondly, to study the effect of different types of dietary fibre
(resistant starch and different non-starch polysaccharides) on microbiota using FISH
64
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TRIAL IV
Trial II was designed with the aim of studying microbial establishment process
after weaning. Cecal digesta from weaned and suckling pigs was collected and realtime PCR was used to study specific bacterial shifts in lactobacilli and enterobacteria
populations. Also, a fingerprinting method (t-RFLP) was evaluated as a useful
method for studying global changes in cecal bacterial profile.
TRIAL V
In Trial I, real-time PCR was assessed as an alternative method to quantify total
bacterial load, lactobacilli and enterobacteria in pig digesta samples. Results were
compared with those obtained by traditional methods as reference values (selective
culture for lactobacilli and enterobacteria, and direct microscopy for total bacteria).
TRIAL II
To assess these three objectives, five different trials were designed. Results will
be included in chapters 4-8.
INTRODUCTION
Chapter 3
and RFLP. Changes in the fermentation pattern were also studied by measuring shortchain fatty acid concentration in the colon.
LITERATURE
REVIEW
OBJECTIVES
Trial IV was designed to study the effect of commercial additives on pig gut
microbiota of weaned pigs. Avilamycin was used as a positive control and sodium
butyrate and a commercial plant extract mixture were tested as alternatives. Real-time
PCR was used to study changes in total bacteria, lactobacilli and enterobacteria along
the gastrointestinal tract, and RFLP was used to assess changes in bacterial profile.
Microbial activity was also measured by purine bases content, and some specific
bacterial enzymatic activities.
TRIAL I
Finally, Trial V was designed to evaluate the effect of a commercial mannanoligosaccharide and organic zinc, administered alone or in combination, on growth
performance, gut microbiota, gut histology and immune response of weaned pigs.
Real-time PCR was applied to quantify lactobacilli/enterobacteria ratio, and purine
bases and short-chain fatty acids, to measure microbial activity. To evaluate the
immune response, immunoglobulin concentration in plasma and digesta, and the
development of continuous Peyer’s Patch were determined.
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
65
66
INTRODUCTION
TRIAL V
TRIAL IV
TRIAL III
TRIAL II
TRIAL I
QUANTIFICATION OF TOTAL BACTERIA,
ENTEROBACTERIA AND LACTOBACILLI
POPULATIONS IN PIG DIGESTA BY REAL-TIME PCR
LITERATURE
REVIEW
Chapter 4
OBJECTIVES
Trial I
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
INTRODUCTION
Trial I
Recently, molecular methods have shown that the complexity of microbial
communities is much greater than previously thought and that the majority of gut
bacteria are still unknown (Pryde et al., 1999; Leser et al., 2002). This lack of
LITERATURE
REVIEW
4.1. Introduction
knowledge is mostly attributed to the failure of many bacteria to grow in a given
molecular methods could be more sensitive and selective than traditional methods
taking into account that they do not rely on the ability of bacteria to grow. Moreover,
DNA-based methods offer the option of storing samples until their analysis, which
could be an important advantage in field conditions.
OBJECTIVES
culture medium (Langendijk, et al., 1995; Huijsdens et al.,2002). Quantitative
Considering the high complexity of gut microbiota, some authors have tried to
microbiota. Conventionally, the ratio lactobacilli:enterobacteria has been used as a
simple index and an increase in this ratio is related with a higher resistance to
TRIAL I
find particular microbial groups that could serve as an index of a health-promoting
intestinal disorders (Muralidhara, et al., 1977; Reid and Hillman, 1999). Specifically
in the weaning pig, lactobacilli could have a predominant role in controlling
months of life (Tortuero et al., 1995; Nemcova et al., 1999).
The objective of this work was to evaluate the use of real time PCR to quantify
TRIAL II
colibacillosis, which is one of the most common intestinal disorders during the first
total bacteria, lactobacilli and enterobacteria in pig digesta samples.
4.2.1. Sample preparation
Samples of jejunum digesta were obtained from healthy early weaned (20 ± 2
TRIAL III
4.2. Material and methods
days) pigs of approximately 40 days old. Animals received commercial diets and
body weight). For comparison of qPCR, selective culture and DAPI staining, 32
animals from the same herd were sampled. To study the effect of pre-treatment of
samples on microbial counts, 18 animals from a second herd were used. The
TRIAL IV
were sacrificed with an intravenous injection of sodium pentobarbitone (200mg/kg
management, housing, husbandry and slaughtering conditions conformed to the
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TRIAL V
European Union Guidelines.
INTRODUCTION
Chapter 4
For microbiological culture procedures and for DAPI staining a fragment of 10
cm from the distal jejunum was tied, cut-off and kept in ice for further dilution. For
LITERATURE
REVIEW
qPCR counts, one gram of digesta was kept in tubes that contained 3 ml of ethanol as
preservative. Samples were gently mixed with the ethanol and stored at 4 ºC until
analysis. To assess the effect of pre-treatment of the sample on the total bacteria
qPCR counts, approximately 5 g from jejunum digesta were sampled and frozen until
analysis.
OBJECTIVES
4.2.2. Bacteria quantification by traditional methods
For selective culture, digesta samples were serially diluted (wt/vol) in sterile PBS
and plated in selective media. Enterobacteria were enumerated using MacConkey
agar at 37 ºC (24h) (CM-115, Oxoid, Madrid, Spain) and lactobacilli in Rogosa agar
at 37ºC in a 5% CO2 atmosphere (48h) (CM-627, Oxoid).
TRIAL I
Direct quantification of total bacteria was carried out by epifluorescent direct
count method (Hobbie et al. 1977) using 4',6-diamidino-2-phenylindole (DAPI)
staining. One gram of sample was diluted ten times with sterile PBS, and 0.5 ml of
this suspension was fixed with 4.5 ml of 2 % formaldehyde. Samples were stained
with DAPI (10 min, 1 μg/ml) and filtered through polycarbonate membrane filters
TRIAL II
(0.22 μm, Whatman International, Kent, UK). Bacteria were enumerated using an
ocular graticule and ten random fields per sample were counted. (Olympus NCWHK
10x, Olympus, Barcelona, Spain).
TRIAL III
4.2.3. Bacteria quantification by real-time PCR (qPCR)
DNA extraction. The equivalent volume to 400mg of digesta samples preserved in
ethanol was precipitated by centrifugation (13000g, 5 min). The DNA from the
precipitate was extracted and purified using the QIAamp DNA Stool Mini Kit
(Qiagen, West Sussex, UK). The lysis temperature was increased to 90 ºC and an
incubation with lysozyme was added (10 mg/mL, 37 ºC, 30 min) to improve the
TRIAL IV
bacterial cell rupture. The DNA obtained was stored at -80º C.
To evaluate possible disregard of bacteria attached to particulate material during
pre-treatment of the samples for culturing and DAPI staining, DNA extraction was
also performed after a previous 1/10 dilution of the samples. One gram of each
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69
sample was diluted ten times with sterile PBS and homogenized 1 minute with a
INTRODUCTION
Trial I
and 4 ml of the liquid phase were centrifugated (20,000 x g, 20 min). The DNA was
extracted and purified from the pellet using the same commercial QIAamp DNA
Stool Mini Kit and procedures described above.
LITERATURE
REVIEW
vortex mixer. Diluted samples were let to stand on the bench during another minute
The DNA from pure cultures of Lactobacillus acidophilus (CECT 903NT) and
centrifugation of 6 ml of culture using the same Qiagen Kit. Pig genomic DNA was
obtained from blood samples that were collected aseptically using the Mammalian
Genomic DNA extraction kit (CAMGEN, Cambridge Molecular Technologies Ltd.,
Cambridge, UK).
OBJECTIVES
Escherichia coli (CECT 515NT) was harvested from the bacterial pellet obtained by
different
primers
were
used:
F-tot
(forward)
5’GCAGGCCTAACACATGCAAGTC3’ (adapted from Marchesi et al. (1998) and
TRIAL I
Quantitative PCR. To quantify total bacteria, lactobacilli and enterobacteria
R-tot (reverse) 5’CTGCTGCCTCCCGTAGGAGT 3’ (adapted from Amann et al.
(1995)
for
total
bacteria.
For
lactobacilli:
F-lac
(adapted
from
Walter
el
al.
(2001))
and
for
enterobacteria
F-ent
5’ATGGCTGTCGTCAGCTCGT3’ (adapted from Leser et al. (2002)) and R-ent
5’CCTACTTCTTTTGCAACCCACTC3’ (adapted from Sghir et al. (2000)). The
TRIAL II
5’GCAGCAGTAGGGAATCTTCCA3’, R-lac 5’GCATTYCACCGCTACACATG3’
oligonucleotides were adapted from published specific primers or probes using the
USA). The different primers were also checked for their specificity using the database
similarity search program nucleotide-nucleotide BLAST (Altschul et al., 1990) and
the absence of amplification of porcine DNA was tested empirically by PCR using
TRIAL III
Primer Express Software to qPCR recommendations (Applied Byosistems, CA,
the DNA extracted from pig blood.
Standard curves were constructed using PCR product of the 16S rRNA gene of E.
et al. (2002). The PCR product was purified with the commercial kit DNA
purification system (Promega Biotech Ibérica, Spain) and the concentration measured
TRIAL IV
coli and L. acidophilus. Primers and PCR conditions were those published by Leser
at 260 nm (Biophotometer, Eppendorf Ibérica S.L., Spain). Products obtained were
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also sequenced (ABI 3100 Genetic Analyzer, PE Biosystems, Warrington, UK) to
INTRODUCTION
Chapter 4
confirm them, and number of copies calculated. Serial dilutions were performed and
102, 103, 104 and 105 copies of the gene per reaction were used for calibration.
LITERATURE
REVIEW
Amplicons from E .coli were used for quantification of the total bacteria and
enterobacteria and amplicons from L. acidophilus for quantification of lactobacilli.
The functions describing the relationship between Ct (threshold cycle) and x (log
copy number) for the different assays were: Ct = -3.19 x + 53.66; R2 = 0.99 for total
bacteria; Ct = - 2.60 x + 46.82; R2 = 0.99 for lactobacilli; and Ct = - 2.32 x + 43.88;
R2 = 0.99 for enterobacteria.
OBJECTIVES
Real-time PCR was performed with the ABI 7900 HT Sequence Detection
System (PE Biosystems, Warrington, UK) using optical grade 96-well plates. The
PCR reaction was performed on a total volume of 25 μl using the SYBR® Green
PCR Core Reagents kit (PE Biosystems). Each reaction included 2.5 μl 10x SYBR
Green buffer, 3 μl MgCl2 (25 mM), 2 μl dNTPs (2.5 mM), 0.25 μl AmpErase
TRIAL I
UNG® (1 U/μl), 0.125 μl AmpliTaq Gold® (5 U/μl), 1 μl of each primer (12.5 μM)
and 2 μl of DNA samples (diluted 1/10). The reaction conditions for amplification of
DNA were 50 ºC for 2 min, 95 ºC for 10 min, 40 cycles of 95 ºC for 15 s, and 60 ºC
for 1 min. To determine the specificity of amplification, analysis of product melting
curve was performed after the last cycle of each amplification.
TRIAL II
4.3. Results and discussion
Minimum levels of detection for the different PCR reactions ranged from 105-106
gene copies/g fresh matter (FM) and were conditioned by the minimum dilution of
sample DNA that did not inhibit the PCR reaction, and by the presence of
TRIAL III
contaminating E. coli DNA in the commercially supplied reagents. Dilution 1/10 was
found not to affect the efficiency of amplification, giving equivalent values to 1/100
and 1/1000 dilutions. On the other hand the degree of contamination of the reagents
was variable but ranged between 10 and 200 copies / reaction. Similar contamination
has been previously described (Suzuki et al., 2000; Nadkarni et al., 2002).
TRIAL IV
Results for total bacteria, lactobacilli and enterobacteria in jejunum samples using
qPCR and traditional methods are shown in Figure 4.1. The values obtained, by
qPCR and traditional methods respectively, were 11.1 ± 0.88 log gene copies / g FM
and 7.8 ± 0.37 log bacteria /g FM for total bacteria; 10.8 ± 1.66 log gene copies / g
FM and 7.9 ± 0.79 log bacteria /g FM for lactobacilli and 8.4 ± 0.56 log gene copies /
TRIAL V
71
g FM and 4.8 ± 1.68 log bacteria /g FM for enterobacteria. It should be noted that
INTRODUCTION
Trial I
confirming Lactobacillus spp. as one of the major groups in upper gastrointestinal
tract of pigs (Khaddour et al., 1998; Reid and Hillman, 1999). In all cases,
quantification by qPCR gave higher values in terms of 16S rDNA than DAPI counts
or CFU (3.4 ± 0.71, 2.9 ± 1.73 and 3.6 ± 1.72 log units higher for total bacteria,
LITERATURE
REVIEW
regardless of the method used, lactobacilli counts were close to total bacteria counts,
ratio (expressed as the difference of logarithms) was similar between methodologies
(2.5 ± 0.58 for PCR and 3.1 ± 0.71 for selective culture, P
= 0.39). Similar
discrepancies between PCR and culturing have been found by other authors
(Nadkarni et al., 2002; Huijsdens et al.,2002) and they have been related to the
OBJECTIVES
lactobacilli and enterobacteria respectively). However, lactobacilli:enterobacteria
multiplicity of 16S rRNA gene copies (Fogel et al., 1999), to the presence of non
viable, or viable but not culturable bacterial cells, and to free DNA. In that sense,
samples were dead and thus, permanently beyond any culture method.
TRIAL I
recently, Apajalahti et al. (2003) found that between 17-34% of bacteria in fecal
The use of real-time PCR with SYBR® Green dye could also lead to
overestimation due to formation of non-specific amplicons (Hein et al., 2001).
However, the dissociation curve obtained at the end of each PCR was checked and
peak, indicating the absence of non-desired PCR products.
Another reason to the overestimation registered, are differences in the pre-
TRIAL II
always had a similar melting point to the standard samples, without any additional
treatment of the digesta. The presence of a quantitative important bacterial
community attached to the coarse particulate material could have been discharged
samples were directly extracted from the original material without any previous
isolation of the bacterial pellet, whereas for culture or DAPI, a previous 1/10 dilution
TRIAL III
somehow with culturing and DAPI methods but not with qPCR. In this study DNA
was performed with a subsequent sub-sampling that generally overlooks most of the
coarse digestive material that persists in the bottom of the tubes. To validate this
from digesta samples or from pre-diluted samples. Results confirmed a reduction in
numbers when subjecting samples to a previous dilution. Mean values were 11.1 ±
0.60 for directly extracted and 10.3 ± 0.51 log units for diluted samples (n = 18). This
TRIAL IV
hypothesis we compared qPCR results for total bacteria using DNA extracted directly
would suggest that a high percentage of microbial population remains attached to the
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coarse particulate material. Previous works have described a high percentage of
INTRODUCTION
Chapter 4
microbes attached to the solid phase (over 70 % in the rumen, Yang et al., 2001).
Moreover, for fecal and digesta samples, DNA extraction protocols are diverse
LITERATURE
REVIEW
(Anderson et al., 2003), some authors extract DNA directly from the samples, while
others isolate previously the bacterial pellet. This previous isolation could affect
results quantitatively and also compromise the representativity of the species
composition taking into account ecological differences between free bacteria and
attached populations (Michalet-Doreau et al., 2001). Results obtained indicate the
importance of previous treatment of samples whatever the method of microbial
OBJECTIVES
quantification we use.
Figure 4.1. Bacterial loads in jejunum digesta of pigs (n = 32) as total bacteria,
lactobacilli or enterobacteria measured by qPCR (log 16S rRNA gene copy number/g
fresh matter (FM)), DAPI staining (log cells/g FM, for total bacteria) or selective
TRIAL I
culture technique (log CFU/g FM, for lactobacilli and enterobacteria). Graph shows
means and standard error of the means.
TRIAL II
TRIAL III
TRIAL IV
In spite of PCR overestimation of microbial counts, values obtained by qPCR and
DAPI for total bacteria showed a significant correlation despite (r = 0.7; P < 0.001)
TRIAL V
73
(Fig 2). It is interesting to point out that qPCR overestimation was higher with the
INTRODUCTION
Trial I
amount of cellular debris and free bacterial DNA with the highest counts or also to an
increase in the percentage of bacteria attached to particulate material that had been
somehow discarded with the DAPI method as we have mentioned before. Another
possible reason to consider is a change in the number of 16S rRNA copies related to
LITERATURE
REVIEW
highest counts than with the lowest counts. It could be due to an increase in the
changes in bacterial species and in metabolic activity of bacteria (Fogel et al. 1999).
significant correlation (r = 0.48; P < 0.01) as did the lactobacilli:enterobacteria ratio
(r = 0.51; P < 0.01). However results obtained for enterobacteria did not show
significant correlation. It could be due to differences in the bacteria species
OBJECTIVES
Similarly to total bacteria, PCR and culture counts for lactobacilli showed a
considered by the two methodologies following a phenotypic (culture) or a genotypic
Figure 4.2. Correlation between the number of total bacteria measured by qPCR
TRIAL I
(qPCR) criterion.
as log 16S rRNA gene copy number/g FM or by DAPI staining as log cell/g FM in
74
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TRIAL IV
TRIAL III
TRIAL II
jejunum digesta samples collected from jejunum samples of pigs.
INTRODUCTION
Chapter 4
4.4. Conclusion
LITERATURE
REVIEW
The results obtained suggest that real-time PCR may well be a practical method
for studying quantitative shifts in pig gut bacteria although numerical values are
higher than for traditional methods. Differences in absolute values could be related to
the amplification of DNA from dead cells with qPCR and to the loss of some particleattached bacteria with DAPI and selective culture. Relative values between groups
OBJECTIVES
such as the lactobacilli:enterobacteria ratio could be used as an index of the gut health
status of pigs. The ease and rapidity of qPCR (once implemented) compared with
traditional culture, and the possibility of storing samples until analysis, could turn
qPCR into the preferred method for quantifying gut bacterial shifts in the near future.
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
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INTRODUCTION
TRIAL V
TRIAL IV
TRIAL III
TRIAL II
TRIAL I
INFLUENCE OF WEANING ON CAECAL MICROBIOTA
OF PIGS: USE OF REAL-TIME PCR AND T-RFLP
LITERATURE
REVIEW
Chapter 5
OBJECTIVES
Trial II
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
INTRODUCTION
Trial II
At weaning, the piglet is subjected to countless of stressors due to complex social,
nutritional and environmental changes which can also enhance piglet susceptibility to
diseases and reduce growth (Pluske et al., 1997; Jensen, 1998). In fact, one of the
most important problems in pig production nowadays is the post-weaning syndrome
LITERATURE
REVIEW
5.1. Introduction
that appears at weaning and involves diarrhoea, growth stasis and consequently
control the activity of the gut microbiota at weaning, antibiotics growth promoters
have been traditionally fed to pigs. However, since their recent total ban in the
European Union (January 2006), new feed strategies and/ or feed additives to
OBJECTIVES
economical loses (McCracken et al., 1995; 1999). In order to enhance growth, and to
maintain piglet gut health are required.
It is it generally recognised that the establishment of a diverse bacterial
plays a key role in the maintenance of the gastrointestinal health avoiding the
colonization by pathogens (Van Kessel et al., 2004). It may be of extreme importance
TRIAL I
microbiota, characteristic and dynamic for each individual (Simpson et al., 2000),
especially at stressful periods such as weaning when sow’s milk withdrawal involves
the removal of immunoglobulin A and lysozyme, among other products, such as
2002). All this, together with the fact that piglets are firstly exposed to many different
complex carbohydrates, causes substantial quantitative and qualitative changes in the
TRIAL II
lactoferrin, that prevent the growth of opportunistic bacteria (Edwards and Parret,
bacterial community (Katouli et al., 1995, 1999; Melin et al., 1997, 2000) , becoming
the piglet more susceptible to microbial disbiosis with potential overgrowth of
little is known about the specific changes of microbial ecosystem during this critical
phase. In that sense, a more exhaustive knowledge of the bacterial shifts that takes
place at weaning would be of great help to follow the proper strategy to replace
TRIAL III
opportunistic disease-causing pathogenic bacteria (Mathew et al., 1996). However,
antibiotics growth promoters in weaned pigs.
Bearing this in mind, an experiment was designed to study microbial shift in pigs
78
TRIAL V
TRIAL IV
at weaning using t-RFLP and real-time PCR.
INTRODUCTION
Chapter 5
5.2. Material and methods
LITERATURE
REVIEW
5.2.1. Animals and housing
A total of 12 piglets (4.4 ± 0.36 kg; 20 ± 2 days, mixed males and females) were
selected from six commercial litters, taking initial body weight into account. One
piglet from each litter was weaned and fed a high quality commercial post-weaning
OBJECTIVES
diet for one week (Table 5.1) whereas the other piglet remained during this week in
the original commercial farm with the dam and the rest of littermates. Weaned pigs
were allocated in a box in the Universitat Autònoma de Barcelona facilities. The
management, housing, husbandry and slaughtering conditions conformed to the
European Union Guidelines.
TRIAL I
Table 5.1. Control diet composition (as fed basis).
Ingredient
%
TRIAL II
TRIAL III
Corn
46.70
Full fat extruded soybeans
17.00
Lactose
15.00
Soybean meal,
10.00
Potato protein
3.77
Whey powder
1.52
L-Lysine HCl (78)
0.17
DL-Methionine
0.10
L-Threonine
0.01
Bicalcium phosphate
3.04
Salt
0.80
Calcium carbonate
0.44
Vit-Mineral premix
0.25
Sepiolite
1.20
TRIAL IV
5.2.2. Sacrifice and sampling
On day 28 of life, the animals were euthanized in the corresponding farm with an
intravenous injection of sodium pentobarbitone (Dolethal, Vetoquinol, S.A., Spain;
TRIAL V
79
200 mg/kg BW). Animals were bled, the abdomen was immediately opened and
INTRODUCTION
Trial II
ethanol (96 %) as a preservative.
DNA extraction. The equivalent volume to 400 mg of digesta samples preserved
in ethanol was precipitated by centrifugation (13000g x 5 min) and DNA from the
LITERATURE
REVIEW
samples (1 g) of the caecum content were taken and kept in tubes with 3 mL of
precipitate was extracted and purified using the commercial QIAamp DNA Stool
increased to 90 ºC and a posterior incubation step with lysozyme was added (10
mg/mL, 37 ºC, 30 min) in order to improve the bacterial cell rupture. The DNA was
stored at -80º C until analysis.
OBJECTIVES
Mini Kit (Qiagen, West Sussex, UK). The recommended lysis temperature was
Real-time PCR (qPCR). Total bacteria, lactobacilli and enterobacteria were
Castillo et al. (2006). The oligonucleotides used were based on regions of identity
within 16S rDNA gene and were adapted from published specific primers or probes
TRIAL I
quantified using real-time PCR following procedures and primers described by
using the Primer Express Software (Applied Byosistems, CA, USA). For total
bacteria,
primers
used
were:
F-tot
(forward)
R-tot (reverse) 5’CTGCTGCCTCCCGTAGGAGT 3’ (adapted from Amann et al.
(1995). For lactobacilli: F-lac 5’GCAGCAGTAGGGAATCTTCCA3’ and R-lac
TRIAL II
5’GCAGGCCTAACACATGCAAGTC3’ (adapted from Marchesi et al. (1998) and
5’GCATTYCACCGCTACACATG3’ (adapted from Walter el al. (2001)) and for
enterobacteria F-ent 5’ATGGCTGTCGTCAGCTCGT3’ (adapted from Leser et al.
al. (2000)). Amplification and detection of DNA by quantitative real-time PCR was
performed with the ABI 7900 HT Sequence Detection System using optical grade 96well plates and SYBR Green dye (PE Biosystems, Warrington, UK). For absolute
TRIAL III
(2002)) and R-ent 5’CCTACTTCTTTTGCAACCCACTC3’ (adapted from Sghir et
quantification, PCR products obtained from the amplification of the whole 16S rDNA
of Escherichia coli (CECT 515NT) and Lactobacillus acidophilus (CECT 903NT)
those published by Leser et al. (2002). The amplified gene from E. coli was used for
absolute quantification of the total bacteria and enterobacteria and the amplified gene
TRIAL IV
were used to construct the standard curves. The PCR conditions corresponded to
80
TRIAL V
from L. acidophilus for quantification of the lactobacilli.
INTRODUCTION
Chapter 5
Terminal-Restriction Fragment Length Polymorfism (t-RFLP). T-RFLP
analysis of bacteria community was performed following the procedure described by
LITERATURE
REVIEW
Höjberg et al., (2005). Briefly, a 1,497 bp fragment of the 16S rDNA gene was
amplified using a 6-carboxy-fluorescein-labeled forward primer: S-D-Bact-0008-a-S20 (5’-6-FAM-AGAGTTTGATCMTGGCTCAG-3’) and reverse primer PH1552 (5’AAGGAGGTGATCCAGCCGCA-3’). Duplicate PCR were made for each sample.
The fluorescently labeled PCR products were purified on QIAquick PCR purification
kit columns (Qiagen, West Sussex, UK,) and eluted in a final volume of 30 μL of
OBJECTIVES
milli-Q water. After that, the resultant PCR product was submitted to a restriction
with Hha I (20,000 U/μl) (Biolabs Inc. New England, USA). The fluorescently
labeled terminal restriction fragments (TRF) were analyzed by capillary
electrophoresis on an automatic sequence analyzer (ABI 3100 Genetic Analyzer, PE
Biosystems, Warrington, UK) in Gene-Scan mode with 25-U detection threshold.
Determinations of the sizes of TRFs in the range of 50 to 700 base pairs were
TRIAL I
performed with the size standard GS-1000-ROX (PE Biosystems).
Treatment of t-RFLP data. Sample data consisted of size (base pairs) and peak
area for each TRF. To standardize the DNA loaded on the capillary, the sum of all
TRF peak areas in the pattern was used to normalize peak detection threshold in each
TRIAL II
sample. Following Kitts (2001), a new threshold value was obtained by multiplying a
pattern’s relative DNA ratio (the ratio of total peak area in the pattern to the total area
in the sample with the smallest total peak area) by 323 area units (the area of the
smallest peak at the 25 detection threshold in the sample with the smallest total peak
area). For each sample, peaks with lower area were deleted from the data set. New
TRIAL III
total area was obtained by the sum of all the remained peak areas in each pattern.
Diversity was considered as the number of peaks in each sample once
standardized. For pairwise comparisons of the profiles, Dyce coeficient was
calculated and dendograms constructed using Fingerprinting II (Informatix, Bio-Rad,
Ca, USA) software and unweighted pair group method with averaging algorithm
(UPGMA).
TRIAL IV
In order to infer the potential bacterial composition in the samples, in silico
restriction for the major pig gut bacteria with the primers and the enzyme used were
obtained using the analysis function TAP-tRFLP from the Ribosomal Database
Project II software (Cole et al., 2003; Table 5.2).
TRIAL V
81
Table 5.2. Theoretical restriction 5’- fragment length predicted for the major pig gut bacteria. Results were obtained from the TAPRFLP tool of the Ribosomal Database II Project software.
Bacteria groups
Lactic Acid Bacteria
Bacteroides and relatives
Fibrobacter
Clostridium and relatives
Proteobacteria
Compatible bacteria a
L. acidophillus, L. brevis, L. bifermentum,
rhamnosum, casei
L. delbruekii sp. Delbruekii
L. delbruekii sp. Lactis
L. fructivorans
Lactococcus lactis, Lactobacillus vaginalis
Enterococcus sp.
Cytophaga
Flexibacter
Bacteroides
Fibrobacter succinogenes
Fibrobacter intestinales
Clostridium coccoides
Clostridium butyricum
Eubacterium
Ruminococcus
Clostridium clostridiforme, C. Symbiosum
Roseburia
Butyrivibrio
Other Clostridium spp.
Escherichia sp
Other enteric bacteria (Salmonella,
Citrobacter, Klebsiella)
In silico restriction b
597, 598, 599
254
223
68
61
216, 218, 220
92, 94, 96, 100
82, 84, 90, 94, 96, 97
95, 96, 98, 101, 102, 104
139, 141, 145
148, 152
66
544
188, 190, 192, 194, 203
189
190
192
193
229, 231, 233, 237
371, 372, 373, 374
Real restriction c
Frequency d
Suckling
Weaned
597, 599
254
221-223
68
62
214
1 (1.45)
4 (1.96)
5 (4.59)
3 (0.88)
6 (14.22)
5 (2.18)
2 (0.54)
1 (0.98)
0
3 (0.59)
5 (19.42)
2(1.35)
89-104
6 (4.06)
5 (5.17)
138, 140, 142-145
148-152
66
544
6 (1.80)
6 (3.46)
3 (1.67)
5 (0.86)
4 (2.70)
5 (0.69)
0
0
188-193
2 (1.19)
4 (0.63)
229-232, 237
376-377
6 (1.30)
3 (0.51)
5 (1.36)
1 (0.55)
367, 370, 371, 372, 373,
a
Major pig gut bacteria with a potential compatible fragment found in at least three animals. Other peaks with 58, 59, 69, 111-120, 123, 133, 162, 211, 278 and 279 did not correspond
with any 16S rDNA sequences in the database from the Ribosomal Database Project 8.1 software.
b
In silico restriction was performed using the tap-tRFLP tool from the Ribosomal Database project II.
c
Terminal fragment length obtained after PCR product restriction with Hha I.
d
Number of animals that showed the peak in each experimental group. In braquets abundance of the peak expressed as % of total area. Mean value is calculated only considering the
animals showing the peak.
INTR
ODU
CTIO
N
LITE
RAT
URE
REVI
EW
OBJE
CTIV
ES
TRIA
LI
TRIAL II
INTRODUCTION
Chapter 5
5.2.3. Statistical Analysis
The effect of weaning on total bacteria, lactobacilli, enterobacteria and
LITERATURE
REVIEW
biodiversity was tested with an ANOVA using the GLM procedures of a SAS statistic
package (SAS Inst., Inc. 8.1, Cary, NC). The individual pig was used as the
experimental unit. Statistical significance was accepted at P ≤ 0.05.
5.3. Results and discussion
OBJECTIVES
The animals remained in good health throughout the experiment. Diarrhea was
not detected in any of the pigs, although there was one case of yellowish liquid faeces
(W group). Initial live weight was similar for both groups, with 4.4 ± 0.16 kg for S
and 4.4 ± 0.15 kg for W, and, as expected, at the end of the experimental period body
weight (BW) was higher for piglets that remain with the sow (6.1 ± 0.25 kg and 5.05
± 0.27 kg for S and W respectively, P < 0.001). Expressed as average daily gain
TRIAL I
(ADG), growth rate was higher for suckling than for weaned pigs (0.25 ± 0.02 and
0.10 ± 0.02 kg for S and W respectively, P < 0.001).
5.3.1. Bacterial quantitative change measured by real-time PCR
The total microbial population, lactobacilli and enterobacteria were quantified in
TRIAL II
caecum digesta using qPCR (Figure 5.1).
The total bacteria counts, expressed as log 16S rDNA copies/g fresh matter (FM),
were similar between groups (12.84 and 12.81 log gene copy number/g FM for S and
W respectively). Similar results were found by Franklin et al. (2002) in piglets
weaned at 24 days where total faecal anaerobic counts were maintained after
TRIAL III
weaning.
Lactobacilli and enterobacteria have been traditionally selected as microbial
groups with a particular significance for gut health. The ratio between these two
bacterial groups, firstly proposed by Muralidhara et al. (1977), has been routinely
used as a gut health indicator, being desirable that lactobacilli outnumber
enterobacteria to improve robustness against opportunistic pathogens.
TRIAL IV
As expected, animals fed with dry food showed a numerical decrease in
lactobacilli population (9.70 vs. 9.01 log gene copy number/g FM for S and F group
respectively, P = 0.24) that was accompanied by an increase in enterobacteria (9.97
vs. 10.78 log gene copy number/g FM for S and F group respectively, P = 0.13). The
ratio enterobacteria:lactobacilli, expressed as difference of logarithms, was
TRIAL V
83
significantly higher in weaned pigs reflecting the contrary effect of weaning on
INTRODUCTION
Trial II
respectively (P = 0.05)). Comparable results have been found before, with an inverse
connection between lactobacilli and enterobacteria during the first week post-weaning
(Risley et al., 1992; Jensen, 1998). This response is due to marked decreases in
lactobacilli in parallel with increases in enterobacteria population (Mathew et al.,
LITERATURE
REVIEW
lactobacilli and enterobacteria populations (0.27 and 1.76 for S group and W group
1996; Franklin et al., 2002). In fact, abrupt weaning has been associated with a 100numbers of Escherichia coli (Huis in’t Veld and Havennar, 1993).
Figure 5.1. Bacterial loads in caecum measured by quantitative PCR (log 16S
OBJECTIVES
fold drop in the numbers of lactobacilli in the intestine, and 50-fold increase in the
rDNA gene copies /g FM) in suckling or weaned pigs.
14
10
*
2,0
8
1,5
6
1,0
4
Total
Lactobacilli
Enterobacteria
Ratio E:L
0,0
TRIAL III
0,5
2
0
TRIAL II
2,5
enterobacteria:lactobacilli (log/log)
log 16S rDNA gene copies /g FM
12
TRIAL I
3,0
Suckling
Weaned
The maintenance of lactobacilli population, that is well adapted to utilise substrate
from the milk (Hopwood and Hampson, 2003) may be of a great interest considering
E. coli (Hillman et al., 1995; Tannock et al, 1999) and also modulating an adequate
immune response (Perdigón et al., 2001), specially important during the first stage of
TRIAL IV
its related effects promoting gut health by inhibition of some other bacteria, such as
84
TRIAL V
life.
INTRODUCTION
Chapter 5
5.3.2. Ecological bacterial changes, t-RFLP results
The similarity indexes of the t-RFLP profiles illustrated in form of a dendogram
LITERATURE
REVIEW
are shown in Figure 5.2. It shows microbial profiles of 11 pigs, due to the fact that
one pig did not present digesta in the cecum at sampling. The effect of weaning on
the ecological composition of microbiota was clearly dominating in comparison with
other factors that could have been affected, such as litter or individual effects. This
was reflected in two clearly separate clusters, one for each experimental group. There
was only an exception for one weaned piglet that grouped in the suckling branch of
OBJECTIVES
the dendogram that interesting corresponded to the animal that showed liquid faeces.
Separation of this piglet in the dendogram might reflect the beginning of some kind
of enteric disbiosis in this piglet, although no differences in productive parameters
measured were observed. In fact, watery stools are related with malabsorption
syndrome that usually appears 3-10 days after weaning (Kyriakis et al., 1989). A poor
adaptation to dry feed in this animal might have caused a higher speed in transit time.
TRIAL I
Besides dry feed introduction, social stress suffered at weaning has also been related
with increases in cortisol release and an increased transit time via the sympathetic
nervous system (Pluske et al., 2002). Moreover, is well known that bacterial
colonisation is highly dependent on flow of digesta, being impaired by a high speed
(Stewart et al., 1999). All these factors might have become into a fail of microbial
TRIAL II
ecosystem to adapt to dry food in this animal, remaining therefore a bacterial
community more similar to that of the suckling period.
Weaning is not only a change of diet, it involves a countless of stressors. Dietary
components are drastically changed; lipids are substituted by carbohydrates as the
main source of energy that with the immaturity of the piglet digestive system may
TRIAL III
result into an important fermentable substrate for the intestinal bacteria. Also, there is
a marked change into the microbial environment, to which the animal is exposed, that
before was mainly determined by the sow. All this, with the withdrawal of milk
supply, and therefore diverse functional components such as different glycoproteins
and oligosaccharides (Pluske et al., 1996b) can also have a crucial effect shaping the
profile of the piglet autochthonous microbiota.
TRIAL IV
In that sense, suckling pigs showed a higher similarity between them (54-78%)
than weaned pigs with more heterogeneous microbial profiles (25-76%). This higher
homogeneity found in the microflora of suckling piglets could have been determined
by a mother effect. In this sense, Katouli and co-workers (1997), studying metabolic
fingerprinting of piglet’s microbiota, demonstrated the high effect of the sow being
TRIAL V
85
the determinant factor in microbiota establishment within the piglet’s first days of
INTRODUCTION
Trial II
On the other hand, the higher variability in microbial profiles in weaned group
would also reflect the stress of the pig at weaning, responding each animal
individually.
Biodiversity, measured as the total number of bands was similar between both
LITERATURE
REVIEW
life.
experimental groups (49.34 for S and 53.40 for F respectively, P = 0.22). Different
(Katouli et al., 1997; Jensen-Waern et al., 1998; Melin et al., 2000) showing that
weaning involves a clear disruption in the normal pig microbiota evolution. After
that, there is a process of re-establishment that can take more or less time depending
OBJECTIVES
works have described a marked decrease in biodiversity just after piglet weaning
on a plethora of factors. A higher biodiversity is desirable due to the fact that a lower
diversity implies lower colonization resistance being the piglet more susceptible to
McBain, 1999; Melin et al., 2004). In our case, the pigs fed with dry food were
probably in the process of reestablishment of a new microbial equilibrium and
TRIAL I
intestinal disorders and proliferation of opportunistic pathogens (MacFarlane and
probably, a later sacrifice could have shown a higher biodiversity. In that sense,
Jensen (1998) found that it takes 2 to 3 weeks after weaning before the fermentative
(1993) found that until 3-4 months after weaning the microbiota is still in evolution.
More recently, Inoue and co-workers (2005) found a recovery in piglet bacterial
TRIAL II
capacity of the microbiota in the hindgut has fully developed, moreover Swords et al
biodiversity 25 days post-weaning.
In silico restriction using Ribosomal Database Project II was used to infer
needed to remind that dispersed phylogenetic groups of bacteria may produce T-RFs
of identical size (Liu et al., 1997) and that a single t-RF in a profile may represent
more than one organism in the sample. Results are therefore presented as potential
TRIAL III
potential ecological changes in the samples. Before considering these results it is
compatible bacterial species and always have some of speculative as direct attribution
of specie to one peak is not completely possible.
major pig gut bacterial groups that have been recently described (Leser et al., 2002).
Figure 5.3 shows an example of the electropherogram for one pig of each
TRIAL IV
In our case, we did an attempt to assign compatible bacteria species among those
experimental group with inference of some compatible bacteria. Table 5.2 shows the
relative abundance (as % of total area) of compatible bacteria that were at least
86
TRIAL V
represented in 3 animals.
INTRODUCTION
Chapter 5
Figure 5.2. Dendogram illustrating the effect of weaning in t-RFLP banding
patterns. The dendogram represents results from 11 piglets sacrificed on day 28 of
100
90
80
70
60
50
40
Percentage of similarity
30
LITERATURE
REVIEW
life. The dendogram distances are in percentage of similarity.
W1
W2
OBJECTIVES
W3
Weaned
W4
S1
S2
S3
Suckling
S4
TRIAL I
S5
S6
W5
Weaned
Figure 5.3. Electropherogram produced from Hha I digestion of 16S rDNA PCR
TRIAL II
products from caecum digesta from one suckling pig and one weaned piglet. The size
and intensity of each band were determined by using Genescan software. Arrows
show the most abundant peaks in the samples.
1
TRIAL IV
Fluorescence detected
TRIAL III
2
Suckling
3
4
1
Weaned
3
Base pairs
1 L. vaginalis/L. lactis 3. Fibrobacter intestinalis
2 C. coccoides
4. L. delbruekii
TRIAL V
87
Lactic
acid
bacteria.
Lactobacillus,
Lactococcus,
Streptococcus,
and
INTRODUCTION
Trial II
are also characterized by the formation of lactic acid (Aguirre and Collins, 1993) and
are described as one of the major groups in the pig gastrointestinal tract (Hill et al.,
2005).
Analysis of electropherograms revealed compatible TRFs with different lactic
LITERATURE
REVIEW
Enterococcus, belong to the Firmicutes with low mol% G + C content in DNA, that
acid bacteria including L. adidophilus, L. bifermentum, L. brevis, L. casei, L.
delbruekii, and L. fructivorans. It is also interesting to remark the fragment of 62
base pairs compatible with both L. lactis and L. vaginalis that was present in all the
animals with a mean contribution around 15 – 20% of total area (see Figure 5.3).
OBJECTIVES
rhamnosum, L. vaginalis, Lactococcus lactis, L. delbruekii sp. lactis, L. delbruekii sp.
Mean area for total lactobacilli was similar between both groups (23.1 % and 21.5 %
for suckling and weaned pigs respectively).although suckling pigs showed higher
Particularly L. delbruekii sp. lactis was present in five animals from this group,
representing near the 5% of total area, whereas no animal of the W group showed any
TRIAL I
biodiversity in compatible TRFs with different lactobacilli species than weaned pigs.
fragment with compatible size. Similarly, L. delbruekii sp. delbruekii was present in
four suckling pigs, and only appeared in one weaned pig.
described before (Krause et al., 1995). It has been related with the different feeding
behaviour of suckling piglets, much more frequent than weaned pigs (Moran, 1982)
TRIAL II
The presence of a higher diversity of lactobacilli in suckling pigs has been
that usually refrain from eating (Le Dividich and Herpin, 1994). This might result in a
higher and continous amount of substrate available for fermentation in the upper
results into the formation of milk clots in the stomach that could act somehow as a
carrier niche for lactobacilli from stomach to small intestine. All these factors could
result in a more beneficial environment for the growth of lactobacilli in the upper
TRIAL III
gastrointestinal tract in suckling pigs. Moreover, the casein component of sow’s milk
gastrointestinal in suckling pigs tract that could be behind the greater diversity of
lactobacilli species observed in posterior sections.
may be compatible with this group (2.18 %) that only appear in two pigs from the
weaned group (1.35%). In the same way, this bacteria group is often described in
TRIAL IV
In the case of Enterococcus sp., five of the six suckling pigs showed a peak that
newborn babies (Favier et al., 2002) and is also in agree with Jensen (1998) who
88
TRIAL V
found a decrease in enterococci when piglets were weaned.
INTRODUCTION
Chapter 5
Bacteroides and relatives. Different species from the phylogenetic group
Cytophaga-Flexibacter-Bacteroides (CFB) phylum (Gherma and Woese, 1992) can be
LITERATURE
REVIEW
compatible with a series of TRFs of similar size ranging from 89 to 104 bp (see Table
5.2). Summed area of these peaks represent 4.0 % of total peak area for suckling and
5.2 % for weaned pigs being therefore the second group in importance behind
lactobacilli.
Savage et al. (1977) demonstrated that Bacteroides spp is on of the predominant
gram negative anaerobes in the adult pig caecum, and have been described also as one
OBJECTIVES
important bacteria in young piglets (Adami and Cavazzoni, 1999) with a marked
increase after weaning (Swords et al., 1993).
Clostridium
and
relatives.
Clostridia
represents a
phenotypically
and
phylogenetically extremely complex and heterogeneous group of organisms.
Sequences of the 16S rRNA have demonstrated deeply branching lineages within the
TRIAL I
clostridia, which included nonclostridial species (Sharp and Ziemer, 1999; Lawson,
1999). All members of the genus Ruminococcus fall within the genus, as well as
Eubacterium species that are scattered throughout the Clostridium genera too (Collins
et al., 1994; Rainey and Janssen, 1995).
In our study, a peak compatible with Clostridium coccoides only appear in three
TRIAL II
suckling pigs representing 1.67 % of total peak area. In the same way, a peak
compatible with C. butyricum (0.86 %) was only found in the suckling group.
Phylogenetically C. butyricum is classified into the Cluster I, Clostridium sensu
stricto, where are found the majority of species of the genera (Collins et al., 1994).
Some other authors agree with our results and have described Clostridium as one
TRIAL III
of the main anaerobic bacteria during suckling period, declining progressively in
abundance with the age (Swords et al., 1993). The presence of C. coccoides may be
considered as beneficial for the piglets due to its production of SCFA. In fact, it has
been used as a probiotic both in animals and humans (Han et al., 1984: Seki et al.,
2003).
Other compatible peaks with different species from the Clostridium clusters I, IV
TRIAL IV
and XIVa and XVIII were found in both groups of animals representing as a mean of
2.49 and 1.99 % of total area for suckling and weaned pigs respectively. However, it
is difficult to conclude potential changes in bacteria belonging to any of those groups
related to weaning process.
TRIAL V
89
Fibrobacter. Compatible peaks with Fibrobacter succinogenes and Fibrobacter
INTRODUCTION
Trial II
suckling and 3.4 % for weaned pigs).
Until recently, these bacteria were grouped into the Bacteroides sub-phylum,
being recently reclassified into the specifically named genus Fibrobacter (Amann et
al., 1990a). Bacteria belonging to this genera are one of the major indigenous
LITERATURE
REVIEW
intestinalis were found in both groups of animals (5.3 % of total peak area for
fibrolytic bacteria in ruminants (Griffiths and Gupta, 2001) although have been also
particular, high numbers of these bacteria have been described in adult pigs (Varel et
al., 1997). Those bacteria show high cellulolityc and hemicellulolityc enzymatic
activities (Gokarn et al., 1997). Therefore, the presence of this both bacteria in both
OBJECTIVES
found in the pig gastrointestinal tract (Varel et al., 1984; Varel and Yen, 1997); in
experimental groups may point out the high potential that the pig have to effectively
Proteobacteria. Proteobacteria phylum includes Enterobacteriaceae family to
which belong different bacteria such as E. coli, Shigella, Klebsiella, Salmonella that
TRIAL I
utilize dietary fibre especially important in adult animals.
have been routinely described as members of the indigenous pig gut microbiota
(Ewing and Cole, 1994). However, we found potential compatible peaks only in four
A bias in the amplification of particular sequences, caused by preferential annealing
of particular primer pairs to certain templates (Suzuki and Giovanonni, 1996) and
TRIAL II
animals, even that we determined enterobacteria counts by qPCR in all the animals.
also the complexity of amplifying bacteria in lower proportions in complex samples
like digesta content might explain absence of compatible TRFs although being
different sequences in the databases are undoubtedly biased by the investigation
interests. In that sense bacterial groups like lactic acid bacteria have received much
attention and huge amount of sequences have been deposited, whereas other groups
TRIAL III
counted by real-time PCR in all the pigs. It is also fair to remark that the presence of
are less represented. This fact might explain, at least partially, the low abundance
found for enterobacteriacea family compared to lactic acid bacteria and inconsistence
Three peaks (161, 173 and 238 base pairs) were found compatible with
TRIAL IV
of T-RFLP with qPCR results..
Mycoplasma arthritidis, Mycobacterium sp. and Staphylococcus sp. respectively.
These three bacteria were not considered in the study taking into account that did not
90
TRIAL V
represent typical pig gut bacteria (Leser et al., 2002).
INTRODUCTION
Chapter 5
5.4. Conclusions
LITERATURE
REVIEW
The results obtained agree with previous works concluding that commercial
weaning produce marked changes in pig caecum microbiota, with an increase in
enterobacteria:lactobacilli ratio after weaning and changes in T-RFLP bacterial
profiles. Even though only presumptions can be made, suckling pigs showed a higher
diversity of compatible TRFs with different lactic acid bacteria than weaned group,
and showed peaks compatible with C. coccoides, and C. butyricum species that were
OBJECTIVES
absent in weaned pigs. In the light of these tentative results, an interesting way to
maintain post-weaning piglet gastrointestinal health at weaning could be to avoid
marked shifts in these characteristic suckling bacteria. In that sense, alternatives to
antibiotics may focus in maintaining as far as possible the weaning microbial profile
at least during the post-weaning transition, by leading to a more favorable
equilibrium. However, more studies are required to increase our knowledge regarding
TRIAL I
the microbiota changes at weaning.
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92
INTRODUCTION
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TRIAL IV
TRIAL III
TRIAL II
TRIAL I
MOLECULAR ANALYSIS OF BACTERIAL COMMUNITIES
ALONG THE PIG GASTROINTESTINAL TRACT
LITERATURE
REVIEW
Chapter 6
OBJECTIVES
Trial III
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
INTRODUCTION
Trial III
The gut microbial ecosystem in mammals is highly complex, typically comprising
more than 400-500 species and viable counts up to 1012 per gram of gut content in the
large intestine (Moore and Holdeman, 1974; Eckburg et al., 2005). Several studies have
investigated the species diversity of the pig intestine through phenotypic analysis of
LITERATURE
REVIEW
6.1. Introduction
isolates obtained by anaerobic culturing (Tannock et al., 1970; Salanitro et al., 1977;
bacteria more readily than others, and is laborious (Zoetendal et al., 2004). The
development of new molecular tools has revolutionized our knowledge of gut microbial
diversity (Pryde et al., 1999; Vaughan et al., 2000). Leser and co-workers (2002)
OBJECTIVES
Robinson et al., 1981; Varel et al., 1987). Culturing however is likely to recover some
sequenced more than 4 200 cloned 16S rDNA sequences from digesta samples of 52
pigs. This work confirmed the complexity of the pig intestinal microbial community
profiling techniques, such as DGGE and T-RFLP that exploit 16S rRNA sequence
differences have also contributed to our understanding of population dynamics along the
TRIAL I
and indicated that much of the pig gut microflora remains uncultured. Meanwhile
different compartments of the gastrointestinal tract of the pig (Simpson et al., 1999).
The availability of 16S rRNA sequences has also facilitated the design of
hybridization (FISH). FISH has the advantage that it avoids potential PCR-bias, while
also giving information about the spatial distribution of gut microorganisms. FISH has
been used to quantify different microbial groups in the human gut (Harmsen et al.,
TRIAL II
phylogenetically targeted oligonucleotide probes that can be used for fluorescent in situ
1999; Hold et al., 2003; Takada et al., 2004) but there is relatively little information on
pig gut microbiology although this technique has been used to study the abundance of
A major incentive to understanding the composition of the gut microflora comes
from the impact of commensal bacteria on host health, in particular pathogen exclusion,
TRIAL III
lactobacilli (Konstantinov et al., 2004b).
immune development and gut metabolism (Stewart et al., 1997). Despite recent
advances, our knowledge of a healthy pig gut microbiology is still far from complete.
environmental changes could help to design strategies to promote health particularly in
young animals. Nutrients that escape digestion in the upper gastrointestinal tract
TRIAL IV
Gaining an understanding of population dynamics and responses to different dietary and
represent the main growth substrate for gut bacteria with complex plant carbohydrates
94
TRIAL V
as their primary available substrates (Salyers et al., 1979). These include soluble non-
INTRODUCTION
Chapter 6
starch polysaccharides (sNSP), insoluble non-starch polysaccharides (iNSP), and
resistant starch (RS) (Englyst and Cummings, 1987). In this way, manipulation of the
LITERATURE
REVIEW
quantity or the type of fibre administered to a pig can be a potential mechanism to
change the structure of the microbial population and make it more resistant to the
establishment of opportunistic pathogens.
The aim of this project was, firstly, to study the quantitative importance of major
bacterial groups along the different sections of the pig gastrointestinal tract and
OBJECTIVES
secondly, to evaluate the potential of dietary fibre to modulate the gut microbial
ecosystem.
6.2 Material and methods
6.2.1. Animals and diets
TRIAL I
The experiment was performed at the Experimental Farms of the Universitat
Autònoma de Barcelona and received prior approval from the Animal Protocol Review
Committee of this Institution. The management, housing, husbandry and slaughtering
conditions were conformed to the European Union Guidelines.
A total of 32 pigs (Pietrain x (Large White x Landrace) of 15 ± 0.38 kg of body
weight, were distributed into 32 pens with forced ventilation. Pens were distributed into
TRIAL II
four experimental diets that included a control diet (CT) (54% corn, 15% barley, 28%
soya-44, 0.7 % vegetable oil, 3% vitamins, minerals and aminoacids), a diet enriched in
resistant starch (GC) by substitution of fine-grounded corn of basal diet (2.5 mm) by
coarse-grounded corn (4 mm), a diet enriched in soluble fiber (BP) by partial
substitution of the corn by 8% of sugar beet pulp or a diet enriched in insoluble fiber
TRIAL III
(WB) by partial substitution of corn by 10 % of wheat bran. Animals were fed ad
libitum for 6 weeks.
6.2.2 Sample collection and processing
At the end of the experimental period, twenty animals (5 per treatment) were
TRIAL IV
euthanised with an intravenous injection of sodium pentobarbitone (Dolethal,
Vetoquinol, S.A., Madrid, Spain; 200 mg kg-1 BW). Animals were bled, the abdomen
was immediately opened and samples of the intestinal content were taken.
For FISH analysis, samples of the stomach, distal jejunum, distal colon and rectum
digesta were taken (500 mg).
Immediately after the collection, the samples were
TRIAL V
95
INTRODUCTION
Trial III
homogenised and diluted ten times with PBS. To remove gross material, the samples
prepared 4% paraformaldehyde. To fix the cells, the samples were incubated overnight
4 ºC and finally stored at -80 ºC until use.
Digesta from proximal colon was homogenized and the pH determined. For short
LITERATURE
REVIEW
were centrifuged (700 x g, 3 min) and 1 ml of supernatant was fixed with freshly
chain fatty acids (SCFA) analysis, samples were collected (5 g) and kept frozen (-20
approximately 50 g were taken, frozen and lyophilised until analysis. For DNA analysis,
samples of approximately 1 g of digesta were kept in tubes with 3 ml of ethanol as a
preservative.
OBJECTIVES
ºC). For purine bases analysis (guanine plus adenine) used as microbial marker,
Fluorescent in situ hybridization (FISH). Samples from the stomach, distal
following groups: Total bacteria (Eub 338), Bacteroides/Prevotella group (Bac303),
Ruminococcus flavefaciens (Rfla729), R. bromii (Rbro730), clostridia cluster XIVa
TRIAL I
jejunum, proximal colon and rectum digesta were assessed with probes for the
(Erec482), clostridia cluster IV species related to Faecalibacterium prausnitzii
(Fprau645), clostridia cluster IX (Prop853), Streptococcus/Lactococcus sp. (Str493) and
Lactobacillus/Enterococcus sp. (Lab158) (Table 6.1) following the method described by
Diluted cell suspensions (10μl) were applied to gelatin coated slides and hybridised
with 10 μl of each oligonucleotide probe (50 ng µl-1 stock solution) in 110 μl of
TRIAL II
Harmsen and co-workers (2002).
hybridisation buffer overnight (except for Bac303 probe, which was hybridised for 2 h).
To prevent fading of fluorescence Vectashield (Vector Laboratories, Burlingame,
DMRXA epifluorescence microscope. Twenty five fields were counted for each sample
(in duplicate).
TRIAL III
California) was added to each sample. Fluorescent cells were counted with a Leica
DNA extraction and purification. Digesta samples (400 mg) preserved in ethanol
was extracted and purified using the commercial QIAamp DNA Stool Mini Kit (Qiagen,
West Sussex, UK). The recommended lysis temperature was increased to 90 ºC and a
posterior incubation step with lysozyme was added (10 mg ml-1, 37 ºC, 30 min) in order
TRIAL IV
were precipitated by centrifugation (13 000 g x 5 min) and DNA from the precipitate
to improve the bacterial cell rupture. The DNA was eluted in 200 μl of Qiagen Buffer
96
TRIAL V
AE (Qiagen, West Sussex, UK) and was stored at -20º C. The purified DNA was
INTRODUCTION
Chapter 6
stabilized with the addition of 4 μl of 40 mg ml-1 BSA (Bovine Serum Albumin, SigmaAldrich Química S. A., Madrid) plus 2 μl of ribonuclease A (Sigma-Aldrich).
LITERATURE
REVIEW
PCR-RFLP analysis . To analyze the total bacteria in the proximal colon digesta, a
580 bp fragment of 16S-rDNA gene was amplified from DNA extracts by PCR using
primers specific to conserved sequences flanking variable regions V3, V4 and V5: 5’CTACGGGAGGCAGCAGT-3’ (forward) and 5’- CCGTCWATTCMTTTGAGTTT-
OBJECTIVES
3’ (reverse). Primers and PCR reaction conditions were those described by Lane and coworkers (1991). The reaction was performed using a GeneAmp PCR System 9700 (PE,
Biosystems, Warrington, UK) thermocycler. The DNA amplification conditions were
94 ºC (4 min); 35 cycles of denaturation at 94 ºC (1 min), annealing at 45 ºC (1min)
with an increment of 0.1 ºC per cycle, extension at 72 ºC (1 min 15 s); and a final
extension at 72 ºC (15 min). Following visual confirmation of PCR products with
TRIAL I
agarose gel electrophoresis, four independent enzymatic restrictions were carried out
(AluI, RsaI, HpaII, CfoI (F.Hoffmann-LaRoche Ltd Group, Basel, Switzerland). The
digestions were performed as recommended by the manufacturer, with the appropriate
restriction buffer at 37 ºC for 3 hours. Different fragments were separated using a 2%
high resolution agarose gel.
TRIAL II
The size and the intensity of the bands within each lane of a gel was analyzed by the
Gene Tools software (Syngene, Cambridge, UK) and the degree of microbial
biodiversity was measured as the total number of different bands obtained from the four
independent restriction digestions. For pair-wise comparisons of the banding patterns
and the construction of dendograms, similarity matrices were generated based on the
TRIAL III
Manhattan distance (Kaufmann and Rousseaw, 1990) that takes into account the size
and the height of the bands generated.
Fermentation product analysis. Analysis of SCFA was performed by GLC using
the method of Richardson and co-workers (1989) modified by Jensen and co-workers
(1995). Purine bases (adenine and guanine) in lyophilised digesta samples (40 mg) were
TRIAL IV
determined by HPLC (Makkar and Becker, 1999). For their analysis purine bases were
hydrolyzed from the nucleic acid chain by their incubation with 2 ml 2 M-HClO4 at
100ºC for 1h, including 0.5 ml of 1 mM-allopurinol as an internal standard.
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97
Table 6.1. Sequence of oligonucleotide probes used in this study.
Probe
Sequence (5’→ 3’)
Targeted bacterial group
Reference
Eub338
GCTGCCTCCCGTAGGAGT
Domain bacteria
Amann et al. 1990b
Bac303
CCAATGTGGGGGACCTT
Bacteroides-Prevotella group
Manz et al. 1996
Rfla729
AAAGCCCAGTAAGCCGCC
Ruminococcus flavefaciens-like
Rbro730
TAAAGCCCAGYAGGCCGC
Ruminococcus bromii-like
Erec482
GCTTCTTAGTCAGGTACCG
Clostridium cluster XIVa
Lab158
GGTATTAGCA(C/T)CTGTTTCCA
Lactobacillus-Enterococcus group
Fprau645
CCTCTGCACTACTCAAGAAAAAC
Faecalibacterium prausnitzii group
Suau et al. 1999
Streptococcus and Lactococcus sp.
Franks et al. 1998
Clostridium cluster IX
Walker et al. 2005
Str493
Prop853
Harmsen et al. 2002
GTTAGCCGTCCCTTTCTG
ATTGCGTTAACTCCGGCAC
INTR
ODU
CTIO
N
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LITE
RAT
URE
REVI
EW
TRIAL II
OBJE
CTIV
ES
TRIAL III
Franks et al. 1998
INTRODUCTION
Chapter 6
6.2.3. Statistical Analysis
The effect of the diet on bacterial, biodiversity, SCFA concentration, pH and
LITERATURE
REVIEW
purine bases concentration in a given intestinal segment was tested with an ANOVA
using the GLM procedures of a SAS statistic package (SAS Institute, INC. 8.1, Cary,
NC). Treatment means were assessed with least significant difference test (LSD)
when overall treatment effects were P < 0.05. Statistical significance was accepted at
P < 0.05.
OBJECTIVES
6.3. Results
6.3.1. Microflora structure along the gastrointestinal tract as analyzed by FISH
Samples were analyzed from stomach, jejunum, proximal colon and rectum of
animals maintained on four different diets. Counts were obtained for each sample
TRIAL I
with the broad eubacterial probe, eub338, and with seven non-overlapping, groupspecific probes. These targeted Firmicute bacteria belonging to clostridial clusters
XIVa (Erec482), cluster IX (Prop853) and cluster IV bacteria related to
Faecalibacterium prausnitzii (Fprau645) or to Ruminococcus flavefaciens/bromii
(Rbro730/Rfla729), as well as streptococci (Strc493) Lactobacilli (Lab) and the
TRIAL II
Bacteroides/Prevotella group of Gram-negative bacteria (Bac303). As expected, total
bacteria per gram of digesta measured with Eub338 probe increased from proximal to
distal sections being at least 100-fold higher in proximal colon and rectum (averaging
approximately 4.0 ×1010 and 5.8 ×1010 respectively) than in stomach and jejunum (3.6
×108 and 2.4 ×108 respectively).
Eub338 counts for the stomach averaged around 4 ×108 g-1 over the four diets. In
TRIAL III
this site the little studied clostridial cluster IX group made up a highly significant
fraction (14-41 %) when compared with the total eubacterial count. Streptococci (15 37 %) and lactobacilli
(8-26 %) were also abundant, while bacteria related to
clostridial cluster IV ruminococci were also abundant on diets BP and WB (27 and 11
% respectively). The probe set used on average accounted for 56-93 % of the eub338
TRIAL IV
count in the stomach.
By contrast, in the jejunal samples the probe set coverage was lower than in the
stomach and particularly on CT diet only covered 32 % of the total bacteria. Lactic
acid bacteria measured (streptococci and lactobacilli) were the main groups and both
together exceeded 24 % of the eub338 counts.
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99
In contrast, the rest of groups
INTRODUCTION
Trial III
measured were less important in this gastrointestinal section and were always below
counts were below the detection limits (counts < 2 ×106 g-1).
Bacterial profiles in the proximal colon and rectum were similar, with cluster
XIVa bacteria amounting to 10-19 %, Bacteroides/Prevotella relatives 4.5-10 %,
LITERATURE
REVIEW
the 6% of total counts. As found in the stomach, cluster IV F. prausnitzii relatives
cluster IV F. prausnitzii relatives 1.4-3.6% and cluster IX bacteria 4.7-7.7%
2%, and less than 0.5%, of the eub338 count respectively. These results confirm the
dominance of anaerobic bacteria related to clostridial clusters XIVa and to the
clostridial cluster IV F. prausnitzii relatives in the dense communities of the large
intestine, by comparison with stomach and jejunum. On the other hand, it is also clear
OBJECTIVES
compared to the eub338 count. Streptococcus and Lactobacillus numbers averaged 1-
that a major fraction of the bacterial variation at these sites is still not accounted for
6.3.2. Effects of fibre on microbial composition as estimated by RFLP and
TRIAL I
by the probes used.
fermentation profiles
The level of biodiversity of the proximal colonic microbial ecosystems expressed
differences related to the dietary source of fiber. Animals fed on diet including wheat
bran showed the lowest biodiversity level (28 ± 0.66 number of bands) compared to
control diet (34 ± 1.12), GC (38 ± 2.48) or BP (37 ± 2.15) diets (P = 0.008).
TRIAL II
as number of bands obtained with the four independent enzymatic restrictions showed
Dendograms (Figure 6.1) evidenced that animals receiving WB clustered separately
having the most similar RFLP patterns (95-97 % similarity) followed by CT (91-94
The pH and purine base concentrations of proximal colon samples were similar
between diets (Table 6.2). Total SCFA concentration was higher for diets enriched
TRIAL III
%); GC (86-91 %) and BP (83-95 %) diets.
with additional fibre although differences did not reach statistical significance (CT vs.
rest of diets, P = 0.06). Diets enriched in non-starch polysaccharides (BP and WB)
showed lower molar percentages of branched chain fatty acids (BCFA) (P = 0.10)
100
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TRIAL IV
and valeric acid (P = 0.002).
INTRODUCTION
Chapter 6
Figure 6.1. Dendogram illustrating the percentage of similarity of PCR-RFLP
banding patterns in samples of proximal colon digesta. The dendogram represents
LITERATURE
REVIEW
results from 20 pigs euthanised.
OBJECTIVES
D1
D2
D3
D4
A1
D5
A2
A3
A4
B1
A5
B2
B3
B4
C1
WB diet
CT diet
GC diet
TRIAL I
C2
B5
C3
C4
C5
BP diet
Table 6.2. (A) Fermentation parameters (pH, total SCFA concentration and
purine bases), in the proximal colon contents from pigs receiving experimental diets.
DIETS
TRIAL II
Parameter
pH
-1
PB (µmol g )
-1
SCFA (µmol g )
CT
GC
BP
WB
SEM
P-value
5.85
6.05
5.78
5.92
0.174
0.72
33.0
31.2
34.3
33.0
6.09
0.98
128
141
155
147
19.5
0.17
(B). Major proportions of the different SCFA
TRIAL III
DIETS
TRIAL IV
SCFA (%)
CT
GC
BP
WB
SEM
P-value
Acetate
63.8
59.5
63.1
60.2
4.78
0.40
Propionate
20.2
25.1
24.9
26.3
5.73
0.35
Butyrate
13.3
13.3
10.4
11.9
2.30
0.16
Valerate
2.78
a
b
1.56
b
1.68
0.463
0.002
Branched-SCFA
1.72
0.74
0.75
0.694
0.10
2.10
ab
1.17
CT: control diet; GC: control diet with coarse-grounded corn; BP: control diet enriched in
soluble fiber by addition of 8% of sugar beet bulp and WB: control diet enriched in insoluble
fiber by addition of 10% of wheat bran.
Each mean represents five individual pigs. Least-squares means within a row lacking a
common superscript letter differ (P < 0.05)
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101
INTRODUCTION
Trial III
Table 6.3. Proportions of specific bacterial groupings in different regions of the
Counts as % Eub338
(×109)/ml
Eub338
Bac303
Erec482
Fprau645 Rbro/Rfla
Prop853
Str493
Lab158
Stomach
CT
GC
BP
WB
0.12
2.4
2.6
±0.11
±0.9
±1.7
0.29
1.8
2.8
±0.27
±0.5
±1.1
bd
bd
bd
3.4c
41.1a
20.2
8.0
±1.6
±18.1
±9.4
±17.5
3.3c
17.0b
36.5
26.2
±1.7
±4.4
±41.4
±37.1
26.9a
25.3ab
14.9
21.7
±1.1
±14.6
±5.3
±16.2
10.5b
14.4b
15.5
11.4
±1.5
±1.7
±8.7
±12.8
2.0
3.3
4.9
15.3
±1.1
±3.4
±5.8
±15.3
3.3
3.7
25.7
11.0
±1.5
±2.2
±22.6
±12.4
2.9
2.1
12.9
11.4
±1.1
±2.0
±17.7
±23.6
2.8
1.0
31.1
25.0
±1.3
±0.5
±36.2
±47.1
0.53
2.2
2.1
±0.49
±1.6
±1.0
0.49
1.9
2.1
±0.63
±0.5
±1.0
0.22 ±
4.7
1.6
0.13
±0.4
±0.6
0.37
3.5
1.5
±0.50
±1.2
±0.4
0.25
5.5
2.9
±0.31
±1.9
±1.4
0.12
5.1
2.0
±0.11
±2.5
±0.7
39.6
8.1ab
12.2
2.0ab
8.9
5.1
1.4
0.2
±15.8
±2.0
±1.4
±0.5
±2.4
±1.4
±0.8
±0.1
44.5
6.8b
13.6
1.4c
8.5
5.1
1.2
0.1
±10.0
±0.7
±2.9
±0.3
±1.8
±1.3
±1.1
±0
37.7
8.6a
11.0
2.5a
9.6
4.7
1.5
0.5
±6.8
±1.5
±3.9
±0.4
±1.3
±0.7
±1.3
±0.5
37.7
10.0a
12.1
1.6bc
9.4
5.8
1.0
0.1
±6.4
±0.7
±2.6
±0.3
±1.6
±1.7
±0.3
±0.2
bd
OBJECTIVES
Count
LITERATURE
REVIEW
porcine digestive tract estimated by fluorescent in situ hybridization.
bd
Proximal colon
CT
GC
BP
WB
Rectum
CT
GC
BP
WB
61.3
8.9
10.4
2.4
4.6
5.5
1.2
0.1
±15.6
±1.8
±4.2
±0.7
±1.6
±1.3
±1.1
±0.1
49.6
9.3
16.7
2.1
4.7
6.4
1.8
0.1
±9.5
±1.6
±9.2
±0.5
±1.0
±2.6
±1.5
±0.1
Bd
44.6
8.9
16.9
3.6
3.7
6.1
2.1
±14.0
±2.2
±7.9
±2.1
±1.3
±1.4
±1.0
47.7
4.5
18.7
3.6
5.3
7.7
1.3
0.1
±25.5
±2.3
±8.4
±0.5
±1.3
±4.5
±0.8
±0.1
102
TRIAL II
WB
bd
TRIAL III
BP
bd
TRIAL IV
GC
bd
TRIAL V
CT
TRIAL I
Jejunum
INTRODUCTION
Chapter 6
LITERATURE
REVIEW
CT: control diet; GC: control diet with coarse-grounded corn; BP: control diet enriched in
soluble fiber by addition of 8% of sugar beet bulp and WB: control diet enriched in insoluble
fiber by addition of 10% of wheat bran.
Values are average ± SD. Values sharing the same superscripts did not show a significant
effect of diet (P > 0.05); where no superscripts are shown, there was no significant effect of
diet.
Bd=below detection (counts <2 × 106 g-1).
OBJECTIVES
6.4. Discussion
Molecular analyses of the diversity of the porcine intestinal microflora have
revealed that a high proportion of the resident bacteria do not correspond closely to
known cultured species (Pryde et al., 1999; Vaughan et al., 2000). Together with
similar 16S rRNA-based work from other mammalian gut communities (Harmsen e
al., 2002; Lay et al., 2005) these analyses are helping to provide a valuable array of
TRIAL I
probes and primers that facilitate studies on the distribution of particular phylogenetic
groups within the intestine, and their responses to different dietary regimes.
The present study shows that particular phylogenetic groupings, detected here by
specific FISH probes, preferentially colonize different regions of the gut. In some
cases the observed distribution agrees with expectations based on the characteristics
of cultured representatives, but other findings were unexpected. The group targeted
TRIAL II
by the Erec482 probe represents Firmicute bacteria that belong Clostridium cluster
XIVa, and was significant mainly in the hind gut (proximal colon and rectum) rather
than in the stomach and jejunum. Most isolates of this group from the human intestine
are highly oxygen sensitive, and are presumed to depend on anaerobic conditions
found only in the dense community of the lower gut. A similar distribution was
TRIAL III
observed for F. prausnitzii-related bacteria, which are also strict anaerobes, and
belong to the clostridial cluster IV. These two groups include the main butyrateproducing species from the human gut (Barcenilla et al., 2000). The
Bacteroides/Prevotella group was less abundant in the pig rectum than has been
reported in comparable studies using automated microscopic analysis of human feces
TRIAL IV
(Harmsen et al., 2002) although lower estimates for human feces have been reported
based on Fluorescence Activated Cell Sorting (FACS) analysis (Lay et al., 2005).
Conversely,
the
Lactobacillus
and
Streptococcus
groups,
that
comprise
microaerophilic and facultatively anaerobic species, made up significant proportions
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103
INTRODUCTION
Trial III
of the microflora of the stomach and small intestine, but made a very small
Little is known from cultural studies about the two remaining targeted groups of
bacteria in the pig gut. The probe used here to detect cluster IX bacteria detected
around 5% of total bacteria found in human faeces (Walker et al., 2005) and a similar
LITERATURE
REVIEW
contribution to the microflora of the proximal colon and rectum.
proportion here in pig rectal contents and proximal colon. The targeted group
Megasphaera and Mitsuokella, most of which produce propionate as an end product.
Remarkably, however, this group accounted for 14-41% of total bacteria detected in
the stomach. It seems unlikely that the same species would account for the
populations of cluster IX bacteria found in the very different environments of the
OBJECTIVES
includes a diverse collection of anaerobes, including Selenomonas, Veillonella,
stomach and large intestine, but further investigations will be needed to establish this.
intestine include specialist fibre-degrading, often cellulolytic, bacteria such as R.
flavefaciens, and starch- degrading species such as R. bromii. These species are
TRIAL I
Cluster IV bacteria classified as ruminococci from the rumen or human large
highly oxygen sensitive and their significant populations (8-10%) in the proximal
colon of the pig correspond with what is presumed to be the site of most efficient
populations of Ruminococcus-related organisms in the stomach on diets BP and WB
were quite unexpected. Again, it is a possibility that the stomach representatives
belong to distinct strain/ species. It should also be noted however that the overall
TRIAL II
breakdown of structural plant polysaccharides. On the other hand, the very high
numbers are far lower in the stomach than in rectal contents, and that FISH detection
can detected metabolically inactive or inviable, as well as viable, cells. Thus there is
populations in the stomach, and that ruminococci might be relatively more resistant to
lysis in the stomach than some of the other groups of anaerobic bacteria found in
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some possibility that re-ingestion of faecal material might produce transient
faeces.
Coprophagy has been described in suckling piglets (Swanson and Gleed, 1981)
hypothesized that a high fiber content in form of NSP could stimulate this behaviour,
explaining the higher amounts of ruminococci in the stomach of BP and WB diets.
Increases in dietary cellulose have been demonstrated to increase the amount of faecal
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and may reasonably be expected in animals allocated in small pens. It could be also
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pellets eaten from the cage floor in captive wild water voles (Woodall, 1989).
INTRODUCTION
Chapter 6
The relatively low probe coverage achieved in this work may partly reflect the
fact that most probes had been designed initially against human faecal bacteria.
LITERATURE
REVIEW
Clearly much more work is required to design probes that target some of the lessstudied groups that colonize the pig gut, particularly for sites such as the jejunum.
We were not able to detect marked dietary changes in gastrointestinal microflora
using FISH. It could be due partially to the failure of our set of probes to target all
microbial groups but also to changes in species composition of the different groups
OBJECTIVES
that had not been detected.
The ability of the diets to promote changes in intestinal ecosystem was confirmed
by changes in the fermentation pattern between diets and by modifications in the
RFLP profiles. It is well known that as carbohydrate sources decreased as
fermentable substrate in the large intestine, fermentation becomes more proteolytic
(Piva et al., 1995). This could explain the differences in SCFA profiles observed in
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animals fed diets rich in NSP (BP and WB). Similar decreases in BCFA with the
inclusion of higher amounts of NSP in the diet have been described by other authors
in pigs (Bach Knudsen et al., 1993). All these fermentation products, which mostly
came from the fermentation of amino acids, reflect somehow the higher availability
of carbohydrates as fermentable substrate in those animals receiving diets with
additional amounts of fibre. These changes could come from both, a change in
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species composition and/or in metabolic activity of microflora. Analysis of ecological
composition of colonic community revealed some changes in composition that were
particularly evident with the WB diet. The inclusion of wheat bran decreased the
biodiversity of the ecosystem and also promoted a much more homogeneous
community compared to the other dietary regimes. Wheat bran diet could be
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considered as a diet enriched in insoluble NSP mostly cellulose and hemicellulose.
Similarly to beep pulp or coarse maize, wheat bran provides plenty of substrate to the
bacteria but in this case this substrate is difficult and time-consuming to ferment
(Bach Knudsen et al., 1991) and this could require a specialized bacterial population.
This type of specialization could be the reason for the lower biodiversity and the
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higher homogeneity in the microflora of these animals. Similar results in terms of
biodiversity were found by Högberg and co-workers (2004) comparing the microflora
of pigs receiving diets differing in the amount and solubility of NSP. Animals
receiving diets with high amounts of insoluble NSP showed the lowest biodiversity
defined as number of terminal restriction fragments detected by T-RFLP.
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The stability of a bacterial ecosystem seems to be directly related to its diversity
a key role in its stability avoiding intestinal disorders and proliferation of
opportunistic pathogens (Kühn et al., 1993; Katouli et al., 1999; Macfarlane et al.,
1999). In this regard, beet pulp or coarse maize would appear as better ingredients
LITERATURE
REVIEW
index (Atlas et al., 1984), and a highly diverse microflora has been considered to play
than wheat bran to promote a robust microflora and prevent the proliferation of
terms as desirable, the resistance against pathogen colonization offered by the
intestinal microflora probably will depend not only on the complexity of the
ecosystem but also on many other factors as the particular microbial species presents,
the type of pathogen challenge or on the different characteristics of the digesta
OBJECTIVES
pathogens. However although a higher biodiversity could be considered in broad
promoted by different diets. Further studies are needed to determine which kind of
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fibre is suitable in each situation.
6.5. Conclusions
This study is the first to examine the major bacterial communities along the GIT
Whereas lactic acid-producing bacteria are abundant mainly in the stomach and
jejunum, strict anaerobes such as F. prausnitzii are only present in proximal colon
and rectum. The abundance of the little studied clostridial cluster IX group is revealed
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of pigs using FISH. Important differences are shown between foregut and hindgut.
along the length of the tract, and this group was shown to make a significant
were not reflected in major bacterial groups quantified by FISH, however
fermentation pattern and community profile analyzed by RFLP showed changes
related to fibre. Globally, increased amounts of fibre promoted decreases in
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contribution to the microbiota of the stomach. Dietary changes in fibre composition
fermentation of protein and particularly wheat bran promoted a less diverse and more
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homogenous microflora.
INTRODUCTION
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REVIEW
OBJECTIVES
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INTRODUCTION
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THE RESPONSE OF GASTROINTESTINAL MICROBIOTA
TO THE USE OF AVILAMYCIN, BUTYRATE AND PLANT
EXTRACTS IN EARLY-WEANED PIGS
OBJECTIVES
Chapter 7
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
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INTRODUCTION
Trial IV
Early weaning makes the piglets an easy target for microbial aggressions
(Wallgren and Melin, 2001). To prevent gastrointestinal disorders and to improve the
post-weaning growth rate, feed-grade antibiotics are used regularly. Despite their
general use, their exact mode of action is not entirely clear. Different mechanisms
LITERATURE
REVIEW
7.1. Introduction
a decrease in the production of growth depressing microbial metabolites and in the
competition for nutrients with the host (Anderson et al., 1999; Hardy et al., 2000).
However other mechanisms related to the selection of a healthier microbial
community could also be implicated.
OBJECTIVES
have been proposed. Most of them are based on a reduction in bacterial numbers with
Concerns about bacterial resistance to antibiotics and general food safety issues
promotant effects of AGPs without their potential drawbacks. The addition of
different organic acids to the feed is one of the most widely used alternatives with
effects that have been related to a reduction in the growth of some bacteria (Partanen,
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have encouraged intensive research on new feed additives to maintain the growth
2001). Herbs have been known since ancient times to have antimicrobial,
antioxidative and antifungal properties. Some of these compounds have been reported
pancreatic enzyme secretions or by having a direct bactericidal effect on gut
microflora (Hardy, 2002). Carvacrol from oregano has demonstrated strong
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to improve animal performance due to their stimulating effect on salivation and
antimicrobial properties (Dorman and Deans, 2000), cinamaldehyde from cinnamon
has shown antioxidant and also antimicrobial effects (Mancini-filho et al., 1998), and
experiment reported here aims to evaluate the effect of an antibiotic, an acidifier, and
a plant extract mixture, on the load, metabolic activity and community structure of the
early-weaned pigs’ gastrointestinal microbiota.
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capsaicin from chili stimulates gastric secretions (Platel and Srinivsan, 2000). The
The experiment was performed at the Experimental Farms of the Universitat
Autònoma de Barcelona and received prior approval from the Animal Protocol
Review Committee of this Institution. The management, housing, husbandry and
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7.2. Material and Methods
110
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slaughtering conditions conformed to the European Union Guidelines.
INTRODUCTION
Chapter 7
7.2.1. Animals and Housing
A total of 40 early-weaned pigs (Pietrain x (Large White x Landrace), mixed
LITERATURE
REVIEW
males and females) from a commercial herd were selected from 10 different litters.
No creep feeding was provided during the lactation period. The animals were weaned
between 18 to 22 d of age with an average initial BW of 5.9 ± 0.71 kg and were
housed in the Universitat Autònoma de Barcelona facilities according to their initial
weight in eight pens (five animals per pen). The 40 pigs were allocated in the same
OBJECTIVES
room and separated by solid walls of 60 cm in height with bars into the top up to 80
cm. Each pen had its own feeder and nipple drinker. The weaning room was equipped
with automatic heating and forced ventilation and the temperature was gradually
reduced from 29 to 25 ºC during the experiment.
7.2.2. Dietary treatments and feeding regime
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Four dietary treatments were used. A control diet was formulated (CT; Table 7.1)
to which three different additives were added: 0.04 % avilamycin, (AB; MAXUS,
Elanco Animal Health, Madrid, Spain), 0.3 % sodium butyrate (AC), and 0.03 %
plant extract mixture (XT). The plant extract mixture was standardized as 5% (wt/wt)
carvacrol (Origanum spp.), 3% cinnamaldehyde (Cinnamonum spp.) and 2%
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capsicum oleoresin (Capsicum annum) in an inert fatty carrier that represented the
remaining 90%. Chromic oxide was included as a digestibility marker in all diets
(0.02%). Details of the diet composition are given in Table 7.1. Pigs were fed the
experimental diets ad libitum for 3 wk weeks after weaning and they had free access
to water throughout the experiment.
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Barley
300
Soybean meal, 44% CP
40
Full fat extruded soybeans
40
Soya protein concentrate
60
Fish meal LT
a
50
Dried whey
Acid whey
40
b
150
Wheat gluten
6.8
Sepiolite
10
Dicalcium phosphate
11
L-Lys·HCl
4.4
DL-Met
2.7
L-Thr
1.9
L-Trp
0.4
Choline chloride, 50% choline
2.0
Chromic oxide
1.5
Vitamin and mineral premixc
3.0
Calculated nutrient compositiond
GE, Mcal/kg
4.75
CP, g/kg
183.9
Ether extract, g/kg
51.1
Crude fiber, g/kg
27.8
Ca , g/kg
6.44
P total , g/kg
6.95
P available , g/kg
4.01
Lysine, g/kg
13.87
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276
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Corn
TRIAL II
g/kg
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Ingredient
LITERATURE
REVIEW
Table 7.1. Control diet composition, as fed basis
Fish meal low temperature: product obtained by removing most of the water and some
or all of the oil from fish by heating at low temperature (< 70 ºC) and pressing.
b
Acid whey: product obtained by drying fresh whey (derived during the manufacture of
cheeses) that has been pasteurized.
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a
c
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Provided the following per kilogram of diet: vitamin A, 13,500 IU; vitamin D3, 2000
IU; vitamin E, 80 mg; vitamin K3, 4 mg; thiamine, 3 mg; riboflavin, 8 mg; vitamin B6, 5 mg;
vitamin B12, 40 µg; nicotinic acid, 40 mg; calcium pantothenate, 15 mg; folic acid, 1.3 mg;
INTRODUCTION
Chapter 7
biotin, 150 µg; Fe, 120 mg as iron carbonate; Cu, 175 mg as copper sulfate 5H2O; Zn, 110
mg as zinc oxide; Mn, 65 mg as manganese sulfate; I, 1 mg as potassium iodate; selenium,
0.10 mg as sodium selenite.
LITERATURE
REVIEW
d
Based on composition values from NRC (1998)
7.2.3. Collection Procedures and Measurements
On d 19 and 21, a total of 32 animals (eight per treatment) were euthanized with
an intravenous injection of sodium pentobarbitone (Dolethal, Vetoquinol, S.A.,
OBJECTIVES
Madrid, Spain; 200 mg/kg BW). Each slaughter day an equal number of pigs per
treatment were included. The pigs selected from each pen were those with individual
BW closest to the mean pen weight. Animals were bled, the abdomen was
immediately opened, and samples of the intestinal content were taken.
For DNA analysis, samples of about 1 g of digesta from the stomach, distal
TRIAL I
jejunum, cecum, and the distal colon were kept in weighed tubes with 3 mL of
ethanol as a preservative. Samples were also taken from the jejunum mucous layer: a
segment of 4 cm was longitudinally cut and gently washed with sterile saline
solution. The mucous layer was scraped with a spatula (250 to 500 mg), placed in
weighed capped tubes, and immediately snap-frozen in liquid N. Samples were kept
at -80 ºC until analysis. Digesta samples (approximately 50 g) from the ileum, cecum,
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proximal colon, distal colon, and rectum were taken for purine base analysis. Samples
were frozen and lyophilized until analysis. For the study of microbial enzymatic
activities, samples of 5 g of cecum and distal colon digesta were snap-frozen in liquid
N and kept at -80 ºC until analysis.
DNA Extraction. Digesta samples (400 mg) preserved in ethanol were
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precipitated by centrifugation (13,000 x g for 5 min), and DNA from the precipitate
was extracted and purified using the commercial QIAamp DNA Stool Mini Kit
(Qiagen, West Sussex, UK). The recommended lysis temperature was increased to
90ºC and a posterior incubation step with lysozyme was added (10 mg/mL, 37 ºC, 30
min) in order to improve the bacterial cell rupture. The DNA was eluted in 200 mL of
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Qiagen Buffer AE (Qiagen, West Sussex, UK) and was stored at -80º C. The DNA
from the mucous layer scrapings was harvested using the same commercial kit. The
DNA from pure cultures of Lactobacillus acidophilus (CECT 903NT) and
Escherichia coli (CECT 515NT) was harvested using the same Qiagen Kit. Pig
genomic DNA was obtained from blood samples using the Mammalian Genomic
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DNA extraction kit (CAMGEN, Cambridge Molecular Technologies Ltd.,
Quantitative Polymerase Chain Reaction (qPCR). The primers used to
quantify the different bacterial groups are listed in Table 7.2. The oligonucleotides
were based on regions of identity within 16S rDNA and were adapted from published
LITERATURE
REVIEW
Cambridge, UK).
specific primers or probes using the Primer Express Software (Applied Byosistems,
configurations, the melting temperature, and percentage guanine and cytosine values
within possible primer/probe sets. The different primers were also checked for their
specificity using the database similarity search program nucleotide-nucleotide
OBJECTIVES
CA, USA). This software was used to check for primer-dimer, internal hairpin
BLAST (Altschul et al., 1990) and the absence of amplification of porcine DNA was
tested empirically by PCR using DNA extracted from pig blood.
performed with the ABI 7900 HT Sequence Detection System using optical grade 96well plates and SYBR Green dye (PE Biosystems, Warrington, UK). Duplicate
TRIAL I
Amplification and detection of DNA by quantitative real-time PCR were
samples were routinely used. The PCR reaction was performed in a total volume of
25 μl using the SYBR Green PCR Core Reagents kit (PE Biosystems, Warrington,
μL dNTPs (2.5 mM), 0.25 μL AmpErase UNG (1 U/μL), 0.125 μl AmpliTaq Gold (5
U/μL) (PE Biosystems, Warrington, UK), 1 μL of each primer (12.5 μM), and 2 μL
TRIAL II
UK). Each reaction included 2.5 μl 10x SYBR Green buffer, 3 μL MgCl2 (25 mM), 2
of DNA samples (diluted 1/10). The reaction conditions for amplification of the DNA
were 50ºC for 2 min, 95ºC for 10 min, and 40 cycles at 95ºC for 15 s and 60ºC for 1
analyzed.
For absolute quantification, PCR products obtained from the amplification of the
TRIAL III
min. To determine the specificity of amplification, the product melting curve was
whole 16S rDNA of Escherichia coli (CECT 515NT) and Lactobacillus acidophilus
(CECT 903NT) were used to construct the standard curves, the PCR conditions
coli was used for absolute quantification of the total bacteria and enterobacteria and
an amplified gene from L. acidophilus for quantification of the lactobacilli. The
functions describing the relationship between Ct (threshold cycle) and x (log copy
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corresponded to those published by Leser et al. (2002). An amplified gene from E.
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number) for the different assays were: Ct = -3.19 x + 53.66; R2 = 0.99 for total
INTRODUCTION
Chapter 7
bacteria; Ct = - 2.60 x + 46.82; R2 = 0.99 for lactobacilli; and Ct = - 2.32 x + 43.88;
R2 = 0.99 for enterobacteria.
LITERATURE
REVIEW
PCR-RFLP analysis. To analyze the total bacteria, a fragment of 16S-rDNA
gene was amplified from DNA extracts by PCR using primers specific to conserved
sequences
flanking
variable
regions
CTACGGGAGGCAGCAGT-3’
V3,
V4
(forward)
and
and
V5:
5’5’-
CCGTCWATTCMTTTGAGTTT-3’ (reverse). Primer and PCR reaction conditions
OBJECTIVES
were those described by Lane et al. (1991). The reaction was performed using a
GeneAmp PCR System 9700 (PE, Biosystems, Warrington, UK) thermocycler. The
DNA amplification conditions were 94ºC (4 min); 35 cycles of denaturation at 94ºC
(1 min), annealing at 45ºC (1 min) with an increment of 1ºC per cycle, extension at
72ºC (1 min 15 s); and a final extension at 72ºC (15 min). After visual confirmation
of the PCR products with agarose gel electrophoresis, four independent enzymatic
TRIAL I
restrictions were carried out (AluI, RsaI, HpaII, CfoI (F.Hoffmann-LaRoche Ltd
Group, Basel, Switzerland). The digestions were carried out as recommended by the
manufacturer, with appropriate restriction buffers at the recommended temperature
for 3 h. Different fragments were separated using a 2% high resolution agarose gel.
The size and the intensity of the bands within each lane of a gel were analyzed by
TRIAL II
the Gene Tools software (Syngene, Cambridge, UK), and the degree of microbial
biodiversity was measured as the total number of different bands obtained from the
four independent restriction digestions.
For pairwise comparisons of the banding patterns and the construction of
dendograms, similarity matrices were generated based on the Manhattan distance
TRIAL III
(Kaufmann et al., 1990) that takes into account the size and the intensity of the bands
generated.
Purine Bases Analysis. Purine bases (adenine and guanine) in lyophilized digesta
samples (40 mg) were determined by HPLC (Makkar & Becker, 1999). For their
analysis, purine bases were hydrolyzed from the nucleic acid chain by their
TRIAL IV
incubation with 2 mL 2 M-HClO4 at 100ºC for 1h, including 0.5 mL of 1 mMallopurinol as an internal standard.
Microbial enzymatic activities. The microbial enzymes were extracted from the
digesta contents by hydrolysis of bacterial cells with lysozyme (5 mg/mL, 37ºC, 3h)
following the method described by Silva et al. (1987). After the incubation period,
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INTRODUCTION
Trial IV
samples were centrifuged (23,000 g for 15 min) and the enzymes, from the
the enzymatic extract were determined by assay of reducing sugars released from
purified substrates according to the Nelson-Somogyi method (Ashwell, 1957). The
substrates were suspended in 0.1N sodium phosphate buffer (pH 6.7). The samples
LITERATURE
REVIEW
supernatant were kept frozen (-80ºC) until analysis. Polysaccharidase activities of
(0.05 mL) were incubated (30 min, 40ºC) with 0.45 mL of each substrate solution
from oat spelts (Sigma-Aldrich Química), soluble starch from potato (Panreac,
Barcelona, Spain), and waxy starch from corn (Sigma-Aldrich Química). Activities
against these four substrates were referred to as CMCase, xylanase, amylase, and
amylopectinase respectively. After the incubation period, the reaction was stopped by
OBJECTIVES
containing carboxymethylcellulose (Sigma-Aldrich Química S. A., Madrid), xylan
denaturing enzyme proteins (100 ºC for 10 min) and the amount of reduced sugars
100 µg/mL) were used as a standard curve. The activity of the enzymatic extract was
expressed as μmoles of neutral sugars released per mL of extract per minute and
referred to the purine bases concentration (bacterial enzymatic activity).
TRIAL I
was quantified spectrophotometrically at 600 nm. Dilutions of glucose (0, 25, 50, and
The effect of diet on microbial counts, biodiversity, purine base concentration,
and enzymatic activities in a given intestinal segment was tested with an ANOVA
TRIAL II
7.2.4. Statistical Analysis
using the GLM procedures of a SAS statistic package (SAS Inst., Inc. 8.1, Cary, NC).
The individual pig was used as the experimental unit. When treatment effects were
probability of differences (PDIFF) function adjusted by Tukey-Kramer (SAS Inst.
Inc.). Purine bases concentrations along different intestinal segments in each animal
were analyzed as repeated measures using the PROC MIXED procedure of SAS.
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established (P < 0.05), treatment least square means were separated using the
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Statistical significance was accepted at P < 0.05.
TRIA
L II
TRIA
L III
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Table 7.2. Material and conditions for the quantification of total bacteria, enterobacteria, and lactobacilli
Group
Name
Total
Enterobacteria
a
Sequence (5’→ 3’)
b
Melting Tc
Position in
Amplicon
(Cº)
E. coli gene
length (bp)d
F-Tot
GCAGGCCTAACACATGCAAGTC
61
23
R-Tot
CTGCTGCCTCCCGTAGGAGT
60
337
F-Ent
(G)ATGGCTGTCGTCAGCTCGT
58
1035
315
Reference
Marchesi et al. (1998)
Amann et al. (1995)
Leser et al. (2002)
385
Lactobacilli
R-Ent
CCTACTTCTTTTGCAACCCACTC
58
1419
F-Lac
GCAGCAGTAGGGAATCTTCCA
58
373
R-Lac
GCATTYCACCGCTACACATG
59
721
Sghir et al. (2000)
349
a
Walter et al. (2001).
Oligonucleotides used as primers (F, forward; R, reverse) for the quantification of 16S rDNA genes from the total bacteria (F-Tot, R-Tot),
lactobacilli (F-Lac, R-Lac), and enterobacteria (F-Ent, R-Ent).
b
Italicized bases denote added nucleotides and in brackets deleted nucleotides from previous published primers.
c
Melting temperature estimated by Primer Express Software (Applied Byosistems, CA, USA).
d
Length of PCR product expressed in base pairs.
INTRODUCTION
Chapter 7
7.3. Results
detected in any of the pigs. Animals receiving the different additives tend to had
greater ADG (P = 0.069) at the end of the experimental period (124.7, 177.4, 177.6,
and 165.9 g for CT, AB, AC and XT respectively; E. G. Manzanilla, personal
LITERATURE
REVIEW
The animals remained healthy throughout the experiment and diarrhea was not
7.3.1. Changes in the total microbial counts
The total microbial population was quantified along the whole gastrointestinal
tract using qPCR (Figure 7.1). In the foregut, the counts, expressed as log 16S rDNA
OBJECTIVES
communication).
copies/g fresh matter (FM), increased from 8.0 ± 1.16 in the stomach to 11.1 ± 0.88 in
microbial population intimately attached to the jejunum mucous membrane was also
quantified and although mean values were lower than counts in the lumen (10.2 ±
0.94 log 16S rDNA copy number/g FM) differences did not reach statistical
TRIAL I
the jejunum, showing a considerable increase of more than three log units. The
significance. The cecum and colon digesta showed mean values of 12.4 ± 0.13 and
12.3 ± 0.93 log 16S rDNA copy number/g FM respectively. These values represent an
significant differences in total bacterial loads related to experimental diets were found
in any part of the analyzed gastrointestinal tract.
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increase of more than one log unit compared to the total counts in the jejunum. No
Figure 7.1. Quantitative PCR for total bacteria. (A) The amplification plot of the
standards used to quantify total bacteria: 2.7 x 1011, 2.7 x 1010, 2.7 x 109, and 2.7 x
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ΔRn (magnitude of the signal generated by the PCR conditions).
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108 16S rDNA gene copies/g fresh matter (FM). Threshold cycle was plotted versus
INTRODUCTION
Chapter 7
(B) DNA concentrations (16S rDNA gene copies / g FM) were plotted vs.
threshold cycle value to construct the standard calibration curve.
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
(C) Bacterial loads in the stomach, jejunum, cecum, distal colon digesta, and in
the jejunum mucous layer, measured by quantitative PCR (log 16S rDNA gene copies
/g FM) in early-weaned pigs receiving a control diet (CT) or the same diet with 0.04
% avilamycin (AB); 0.3 % butyric acid (AC) or 0.03 % plant extract mixture (XT).
Bars represent means and standard error of the means.
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Total bacteria, log (16S rDNA copies / g FM)
14
13
CT
AB
AC
XT
12
11
10
9
8
7
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6
Stomach digesta
Jejunum digesta
Jejunum mucous
membrane
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119
Cecum digesta
Distal Colon
digesta
INTRODUCTION
Trial IV
In order to find possible effects of the additives on particular microbial groups, we
also quantified enterobacteria and lactobacilli in the jejunum and cecum using qPCR
(Table 7.3). Both groups increase in number from the jejunum to the cecum; however,
while the enterobacteria showed an increase of around four log units, lactobacilli
LITERATURE
REVIEW
7.3.2. Changes in the microbial ecosystem
increased only by around two logs. Expressing the difference between both bacteria
lower ratio values than the jejunum. Between dietary treatments, XT promoted an
increase in the lactobacilli:enterobacteria ratio in the cecum when compared to the
CT (P = 0.02), which can be explained by an increase in lactobacilli numbers (P =
OBJECTIVES
groups as a ratio of logarithms (lactobacilli:enterobacteria ratio) the cecum showed
0.02). The AC diet also showed higher lactobacilli:enterobacteria ratio mean values;
Table 7.3. Bacterial populations, size of lactobacilli, and enterobacteria in the
distal jejunum and cecum measured by qPCR (log (16S rDNA gene copies / g FM)
TRIAL I
however, differences with control diet were not significant (P = 0.49).
in early-weaned pigsa
Bacteria
CT
AB
AC
XT
SEM
Jejunum
Enterobacteria
8.2
8.6
8.6
8.0
0.20
Lactobacilli
11.5
10.6
10.9
10.2
0.59
lactobacilli:enterobacteriac
3.30
2.02
2.32
2.23
0.583
Enterobacteria
12.4
12.5
12.4
12.4
0.05
Cecum
Lactobacilli
lactobacilli:enterobacteria
a
12.9
z
z
0.48
12.9
z
0.43
z
13.1
yz
0.75
yz
13.5
y
0.11
1.10
y
0.129
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Segment
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Dietsb
Each mean represents eight individual pigs.
b
c
Relation between lactobacilli and enterobacteria populations expressed as ratio of
logarithms.
y,z
Least-squares means within a row lacking a common superscript letter differ (P <
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Experimental diets: CT, control diet; AB, control diet with 0.04 % avilamycin; AC,
control diet with 0.3 % butyrate; and XT, control diet with 0.03 % plant extract mixture.
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0.05).
INTRODUCTION
Chapter 7
In samples of the jejunum digesta, a PCR-amplified region from the microbial
16S rDNA was analyzed using the RFLP method (Figure 7.2). The effect of different
LITERATURE
REVIEW
additives on biodiversity, measured as number of bands, was evident when
considering the decrease in number of bands for the control diet (28.9, 38.5, 38.8, and
32.0 for CT, AB, AC, and XT respectively; P
= 0.03). The effects of the
experimental diets on microbial composition were more clearly distinguished by the
cluster analysis. A dendogram comparing different banding patterns is shown in
Figure 7.2. Distinct clusters according to the different diets were observed. The
OBJECTIVES
acidifier diet promoted the biggest structural changes (63.5% similarity) followed by
the XT diet (66.9 %), and then the AB diet (73.3 %).
Figure 7.2. Ecological changes in microbial population of jejunum digesta
measured by RFLP. (A) Example of gel electrophoresis of the PCR-amplified V3,
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V4, and V5 regions of the 16S rDNA restricted with the enzyme Hpa II (for more
details see materials and methods). Each line represents different animals receiving
the control diet (CT) or the same diet with 0.04 % avilamycin (AB); 0.3 % butyric
acid (AC); or 0.03 % plant extract mixture (XT).
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
121
INTRODUCTION
Trial IV
(B) Dendogram illustrating the correlation between experimental diets in PCRduring the second sampling day (d 21). The dendogram distances are in percentage of
similarity
Percentage of similarity
70
75
80
85
90
95
CT
73.3
OBJECTIVES
65
LITERATURE
REVIEW
RFLP banding patterns. The dendogram represents results from 16 piglets killed
AB
XT
63.5
TRIAL I
66.9
7.3.3. Changes in metabolic bacterial activity
TRIAL II
AC
Total microbial activity along the hindgut was also studied according to the
concentration of all purine bases in the digesta. Evolution of the purine bases
according to the different diets is shown in Figure 7.3. All the treatments show an
increase in PB concentration from the ileum to the cecum that is quantitatively larger
TRIAL III
concentration along the ileum, cecum, proximal colon, distal colon, and rectum,
for the CT and AB diet than for the AC and XT diet. In the hindgut, the evolution of
the PB concentration also shows differences between diets (diet x intestinal section, P
values in previous intestinal sections. Finally, PB concentration in the rectum was
similar for all the treatments.
122
TRIAL V
abrupt decline from the colon to rectum, the rest of the treatments reached maximum
TRIAL IV
= 0.01). While the CT diet reached maximum values at the end of the colon, with an
INTRODUCTION
Chapter 7
In order to detect changes in the microbial metabolic activities between diets,
different carbohydrase microbial activities in the cecum and distal colon digesta were
LITERATURE
REVIEW
also measured. We could not detect enough carboxymethylcellulase or xylanase
activity in most of the samples and therefore data are not shown. However, we did
find detectable amylase and amylopectinase activity, which is shown in Table 7.4.
The data showed high variability, precluding the ability to find any differences
between treatments.
OBJECTIVES
Figure 7.3. Purine bases (PB; adenine and guanine) concentration (μmol/g DM)
in digesta samples from the ileum, cecum, proximal colon, distal colon and rectum in
early-weaned pigs receiving a control diet (CT) or the same diet with 0.04 %
avilamycin (AB); 0.3 % butyric acid (AC); or 0.03 % plant extract mixture (XT). The
asterisks show that diets within an intestinal section differ (P < 0.05). Differences
TRIAL I
also occurred between intestinal sections (P < 0.001) and in relation to diet x
intestinal section interaction (P = 0.01).
60
CT
TRIAL II
AB
PB, μmol/g DM
50
AC
*
*
XT
40
30
TRIAL III
20
*
10
Ileum
Cecum
Proximal
Colon
TRIAL IV
TRIAL V
123
Distal Colon
Rectum
INTRODUCTION
Trial IV
Table 7.4. Bacterial enzymatic activity in samples of the cecum and distal colon
Enzyme
Segment
CT
AB
AC
XT
SEM
Amylase
Cecum
1.11
0.27
0.33
1.49
1.112
Distal Colon
0.67
0.88
0.99
0.85
0.344
Cecum
1.80
1.50
2.08
4.34
1.810
Distal Colon
0.99
1.72
1.64
0.58
0.336
Amylopectinase
a
OBJECTIVES
Dietsb
LITERATURE
REVIEW
contents from early-weaned pigsa
Each mean represents eight individual pigs.
b
TRIAL I
Experimental diets: CT, control diet, AB; control diet with 0.04 % avilamycin, AC;
control diet with 0.3 % butyrate; and XT, control diet with 0.03 % plant extract mixture.
7.4. Discussion
GIT, which was also true for butyrate and the plant extracts. Although it is generally
accepted that antibiotics reduce the number of bacteria in the gut at growth promoting
doses, results obtained in the literature are not always consistent and probably depend
TRIAL II
We found that the antibiotic did not reduce the total microbial counts along the
on the type and doses of antibiotic administered. Collier et al. (2003) using a similar
PCR methodology described a decrease in the total bacteria in the ileum of growing
d 21, however, the effect disappeared after 28 d, which reflects the microbial
community’s adaptive response, replacing susceptible strains with resistant
TRIAL III
pigs after 2 wk of treatment with 40 ppm of tylosin. This decrease was observed until
organisms. In our case, this new equilibrium could have been reached sooner (2 wk),
which would explain the absence of an antibiotic effect on the total bacteria counts.
estimated by qPCR were similar to those described by other authors for culturable
bacteria in pigs of a similar age (Jensen and Jorgensen, 1994; Krause et al., 1995;
TRIAL IV
The values for the total bacteria in the stomach, jejunum, cecum, and colon
McFarland, 1998). It is fair to remark that values were always close to the highest
124
TRIAL V
levels, probably due to the trend of qPCR to overestimate microbial populations.
INTRODUCTION
Chapter 7
Some authors, when comparing qPCR with culture methods, have also described
discrepancies of one or even two log units (Nadkarni et al., 2002; Huisjdens et al.,
LITERATURE
REVIEW
2002). These discrepancies could be explained mainly by the presence of a high
number of viable but not culturable bacterial cells in the digesta samples (RigottierGois et al., 2003), the amplification and later quantification of free DNA from dead
bacteria, and the multiplicity of 16S rDNA genes per genome in prokariotic
organisms (Fogel et al., 1999).
OBJECTIVES
In the light of the absence of significant effects on the total microbial counts, it
seems feasible that antibiotics and other alternatives such as organic acids or plant
extracts, could act not by reducing the total size of the microbial population but by
promoting the selection of particular bacteria. In this respect, the different spectra that
antibiotics have and also the specific susceptibility of bacteria to different organic
acids is well known (Cherrington et al., 1991). Possemiers et al. (2004) using qPCR
TRIAL I
could not detect changes in the total microbial population after adding an antibiotic to
an in vitro simulator of the human microbial ecosystem; however, using group
specific primers they could detect a decrease in the number of bifidobacteria.
Looking for ecological changes, the lactobacilli and enterobacteria populations
were quantified in the jejunum using qPCR. The relationship between these bacterial
TRIAL II
groups has traditionally been considered as an index of desirable or undesirable
bacteria in pigs, relating a high index with a higher resistance to intestinal disorders
(Ewing and Cole, 1994). From the additives tested, XT showed the clearest effect,
increasing the lactobacilli:enterobacteria ratio compared to the control. Increases were
observed in the cecum (P = 0.006) mainly due to an increase in the number of
lactobacilli. Previous results with the same plant extract mixture also showed
TRIAL III
increases in the lactobacilli:enterobacteria ratio in the jejunum of weaned pigs due to
an increase in lactobacilli numbers (Manzanilla et al., 2004). It is difficult to find an
explanation for this promoting effect taking into account that most of the in vitro
studies with plant extracts have shown an unspecific antimicrobial effect (Hammer et
al., 1999). However these consistent results seem to point to some kind of prebiotic
TRIAL IV
effect on the lactobacilli population, either by a direct or indirect effect through an
ecological change in the intestinal microbiota. Adding butyrate to diets also promoted
higher mean values in the lactobacilli:enterobacteria ratio although in this case
differences compared to the control were not significant (P = 0.17). There are few
publications studying the inclusion of n-butyrate in diets for weaned piglets and its
TRIAL V
125
INTRODUCTION
Trial IV
effects on microbial populations. Galfi and Bokori (1990) using 0.17 % sodium ndecrease in the proportion of coliform bacteria with a simultaneous increase in
lactobacilli, however these authors also found an increase in the ileal concentration of
butyrate that we did not observe (data not shown). Van Immersel et al. (2004) using
LITERATURE
REVIEW
butyrate in the diets of weaned piglets observed changes in the ileal microbiota with a
microencapsulated butyric acid in young chickens could also demonstrate a decrease
interesting to note that the same authors using other organic acids like formic and
acetic acid observed the opposite effect with an increase in Salmonella in the cecum
colonization. Probably many other factors, such as the activation or inhibition of
different metabolic routes with different organic acids, are involved in the changes
OBJECTIVES
in Salmonella in the cecum colonization after an experimental infection. It is
observed with the acidifiers and not only a simple effect caused by a lower pH. This
acidifiers on microbial populations described in the literature (Hebeler et al., 2000;
Canibe et al., 2001; Février et al., 2001).
TRIAL I
complexity could explain the diverse and sometimes contradictory effects of different
Avilamycin is an antibiotic mainly active against gram positive bacteria;
therefore, we could expect a decrease in lactobacilli numbers. However, we did not
in bacterial numbers (Decuypere et al., 2002). Similarly, Collier et al. (2003) using
tylosin (another macrolide active against gram positives) could not detect any
decrease in lactobacilli but rather an increase, which is particularly intriguing and
TRIAL II
find this effect. Other authors using avilamycin (50 ppm) also did not find differences
could reflect complex interactions between different species in the bacterial
ecosystem. When using RFLP to analyze variations in the bacterial community, we
point out how profiles for each treatment were clustered separately, the cluster for the
animals that received the AC diet showed the most difference. Differences in the
TRIAL III
evidenced changes in band patterns related to dietary treatments. It is interesting to
RFLP patterns were due to an increase in the biodiversity in the microbial ecosystem
with the use of additives (number of bands) and also to a change in the species
suggested that a more complex microbial community would have a higher robustness
in response to changes in the intestinal environment promoted by different dietary
ingredients or stress and that the beneficial effects of antimicrobial additives could be
TRIAL IV
composition of the community (type of bands). From our results, it could be
related, to an improvement of the adaptive capacity of commensal microbiota as a
126
TRIAL V
natural barrier defense against the overgrowth of pathogens, more than to a reduction
INTRODUCTION
Chapter 7
in bacteria numbers. Other authors using similar fingerprinting techniques
(Denaturant Gradient Gel Electrophoresis (DGGE); McCracken et al., 2001) and
LITERATURE
REVIEW
comparing fecal microbial populations from rats receiving diets supplemented or not
with antibiotics, did not detect changes in the biodiversity but they did detect how
bacterial species that form each microbial community were significantly altered by
the antibiotic. Similarly, Collier et al. (2003) working with pigs receiving 40 ppm of
tylosin for more than 21 d, found that the number of DGGE bands in ileal samples
was similar to the number in the control diet but that the banding patterns were
OBJECTIVES
treatment dependent.
The evolution of purine bases concentration along the hindgut showed that the
main differences were between diets CT and AB. While in the CT diet, the purine
bases concentration reached its maximum value at the distal colon, decreasing
afterwards, the AB diet purine concentration reached its maximum values at the
TRIAL I
cecum. Previous results from our group described similar patterns when comparing
animals receiving diets differing in the amount of resistant starch (Martinez-Puig et
al., 2003). In that case, the purine bases concentration decreased earlier in animals
receiving the diet with a lower amount of fermentable starch. In the present work,
experimental diets have the same ingredient composition except for the added
additives therefore changes in fermentation should not be attributed to differences in
TRIAL II
dietary carbohydrates. However, changes in the extent of digestion and absorption of
nutrients at the foregut level could potentially have promoted the arrival of different
amounts of fermentable material to the hindgut and therefore changes in microbial
carbohydrases activity, but we were unable to observe that occurrence. If additives
did promote differences, they were not big enough to be detected by this
TRIAL III
methodology. It is interesting to point out that while the amylase and amylopectinase
activities were comparable to those described by other authors in growing pigs
(Morales et al., 2002), cellulase or xylanase activities were not detected. This lack of
enzymatic bacteria activity could be related to an insufficient adaptation of
microbiota to digesting complex carbohydrates like cellulose or hemicellulose in
young animals.
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TRIAL V
127
INTRODUCTION
Trial IV
Results suggest that the effects of some growth promoters could be related more
with changes in the species and complexity of the microbiota than to a simple
decrease in bacterial colonization of previous sections of the gastrointestinal tract.
More specific studies are required to clarify how these products modify pig
LITERATURE
REVIEW
7.5. Implications
gastrointestinal bacteria, which would facilitate their most judicious use in field
128
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TRIAL IV
TRIAL III
TRIAL II
TRIAL I
OBJECTIVES
conditions.
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
130
INTRODUCTION
TRIAL V
TRIAL IV
TRIAL III
TRIAL II
TRIAL I
USE OF MANNAN-OLIGOSACCHARIDES AND ZINC
CHELATE AS GROWTH PROMOTERS AND DIARRHEA
PREVENTATIVE IN WEANING PIGS: EFFECTS ON
MICROBIOTA AND GUT FUNCTION
LITERATURE
REVIEW
Chapter 8
OBJECTIVES
Trial V
INTRODUCTION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
INTRODUCTION
Trial V
Reducing post-weaning diarrhea is one of the main challenges for the pig
industry. In commercial practice, the use of different in-feed additives has been
traditionally recommended as a way to help the piglet during this stage. Among these
LITERATURE
REVIEW
8.1. Introduction
agents, mannan-oligosaccharides (MOS) and supplements of zinc have been some of
Mannan-oligosaccharides, Bio-Mos (Alltech Inc, USA; BM) derived from the
outer cell wall of a selected strain of yeast have been extensively used to enhance gut
health (Pettigrew, 2000; Miguel et al. 2004). Research suggests that BM interferes
OBJECTIVES
the proposals with probed positive results.
with bacterial attachment to the epithelial cell (Spring et al., 2000) and can also
enhance immunity (Newman and Newman, 2001; O’Quinn et al., 2001). Zinc oxide
diarrhea during weaning (Poulsen, 1995) and actually, it has become routinely used in
nursery diets as a growth promotant, although its mode of action is not entirely clear.
TRIAL I
(ZnO) at pharmacological levels (2,000 to 3,000 ppm) has been used to prevent
Various studies suggest that it could be mediated by a luminal (Katouli et al., 1999),
an intestinal (Carlson et al., 1999) or a systemic effect (Case and Carlson, 2002).
high levels of ZnO are associated with BM (Davis et al., 2002). However, the use of
high doses of inorganic Zn has raised some environmental concerns due to the
elevated excretion levels in faeces. If the mode of action of Zn is based on a systemic
TRIAL II
Recently, some experimental trials have studied the possible synergistic effect when
effect, then the use of alternative sources of organic forms of Zn, with a higher
release to the environment whilst maintaining the benefits to the animal. Case and
Carlson (2002) demonstrated equivalent efficacy of low doses of organic sources of
Zn compared to pharmacological levels of ZnO under certain conditions.
TRIAL III
bioavailability, would allow a reduction in its feed concentration, and also in its
The main objective of this work was to study the growth promoting effect on
weaning pigs of BM derived form the yeast cell wall of a selected strain of
132
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TRIAL IV
Saccharomyces cerevisiae , and a Zn chelate and the combined use of both additives.
INTRODUCTION
Chapter 8
8.2. Material and methods
LITERATURE
REVIEW
The experiment was carried out in the Experimental Farms of the Universitat
Autònoma de Barcelona in Spain and received prior approval from the Animal
Protocol Review Committee of this Institution.
OBJECTIVES
8.2.1. Animals and diets
A total of 128 early-weaned pigs (Pietrain x (Large White x Landrace, 66 males
and 62 females) were selected from a commercial herd. The animals were weaned
between 18 to 22 days of age with an average initial BW of 6.7 ± 1.17 kg.
The animals were housed into 32 pens (four pigs per pen) taking litter and initial
body weight into account. Animals received four dietary treatments. A control diet
TRIAL I
was formulated (CT) to which 0.2 % of a commercial source of mannanoligosaccharide (Bio-Mos® Alltech Inc, USA; BM), 0.08% organic Zn equivalent to
80 ppm of Zn: (Bioplex Zn™ Alltech Inc, USA; BP’) or both additives (BMP) were
added. No medication or other additives were included in any of the diets.
The experiment lasted for 5 weeks including a pre-starter period of two weeks and
TRIAL II
a starter period of three weeks. During the starter period, diets were slightly modified
according to the requirements of the animals but maintaining constant levels of BM
and organic Zn. The composition of the control diets are showed in Table 8.1. Diets
were formulated following the recommendations of NRC (1998). Animals were fed
ad libitum and had free access to feed and water. At the end of the second week, and
TRIAL III
just before changing to the starter diet, animals were challenged by a controlled
stress. Stress consisted of lowering the room temperature from 27 to 17 ºC and feed
deprival over a period of 10 hours.
8.2.2. Performance and collection procedures
TRIAL IV
Body weight was recorded on a weekly basis and feed intake (by pen) was
recorded daily during the first week and weekly thereafter. Average daily gain (ADG)
and gain:feed data were calculated individually and by group respectively.
TRIAL V
133
Two persons who were blind to treatment modality monitored faecal consistency
INTRODUCTION
Trial V
ranking form 0 to 3 with 0 = normally shaped faeces, 1= shapeless faeces, 2 = soft
faeces and 3 = thin, liquid faeces.
On day 14, 32 animals, one from each pen, were sacrificed with an intravenous
LITERATURE
REVIEW
daily during the first three weeks. Faecal morphology was classified using a scale
injection of sodium pentobarbitone (Dolethal, Vetoquinol, S.A., Madrid, Spain)
whole gastrointestinal tract (GIT) tied and excised. Weight of the whole GIT, full and
empty stomach, and small intestine, empty ileum, and full hindgut were recorded.
Lengths of the whole small intestine and ileum were also registered. Samples for
histology were taken from the distal jejunum and transferred to 10 % neutral buffered
OBJECTIVES
(200mg/kg BW). . Animals were bled, the abdomen was immediately opened and the
formaldehyde.
determined. Samples (approximately 5 g) were kept frozen (-20 ºC) until analysis for
short-chain fatty acids (SCFA). Digesta samples from the ileum, caecum and rectum
TRIAL I
Digesta from the stomach, ileum and caecum was homogenised and pH
were also frozen (-20 ºC) and lyophilised until analysis of purine bases (PB).
Lyophilised ileal samples were also analyzed for protein contents and IgA
DNA extraction and posterior microbiological studies.
TRIAL II
concentration. Samples of jejunum digesta were taken and preserved in ethanol for
8.2.3. Analytical methods
Short-chain fatty acid analysis. Analysis of SCFA was performed by GLC using
Purine Bases analysis. Purine bases (adenine and guanine) in lyophilised digesta
samples (40 mg) were determined by HPLC (Makkar & Becker, 1999). For the
analysis,
TRIAL III
the method of Richardson et al. (1989) modified by Jensen et al. (1995).
purine bases were hydrolysed from the nucleic acid chain by their
incubation with 2 mL 2 M-HClO4 at 100ºC for 1h, including 0.5 mL of 1 mM-
Immunoglobulins. IgA, IgG and IgM concentration in serum was quantified
using Pig IgA, Ig G and IgM ELISA Quantitation Kits. (Bethyl Laboratories, Inc.,
TRIAL IV
allopurinol as an internal standard.
Montgomery, TX). For the determination of IgA in ileum digesta samples, the
134
TRIAL V
method of Swanson et al., (2002) was used. Samples (2g) were lyophilised and
INTRODUCTION
Chapter 8
crushed with a mortar before being placed in an Erlenmeyer flask along with 20 mL
PBS solution, pH 7.2. Samples were mixed for 30 min at room temperature and then
LITERATURE
REVIEW
centrifuged at 20.000 x g for 30 min. The supernatant was collected and ileal Ig
concentrations were determined using the same kits used for serum samples.
Calculation of Ig concentration per crude protein, was determined in ileum digesta as
total N following the Kjeldahl method (AOAC, 1990).
DNA extraction. Digesta samples (400 mg) preserved in ethanol were
OBJECTIVES
precipitated by centrifugation (13000g x 5 min) and DNA from the precipitate was
extracted and purified using the commercial QIAamp DNA Stool Mini Kit (Qiagen,
West Sussex, UK). ). The recommended lysis temperature was increased to 90 ºC and
a posterior incubation step with lysozyme was added (10 mg/mL, 37ºC, 30 min) in
order to improve the bacterial cell rupture. The DNA was eluted in 200 mL of Qiagen
Buffer AE (Qiagen, West Sussex, UK) and was stored at -80º C.
TRIAL I
Quantitative PCR. Microbial populations of enterobacteria and lactobacilli in
ileum digesta samples were quantified by real time PCR using SyBR Green following
Castillo et al. (2006) specifications.
Histological analysis. Formalin-fixed samples were included in paraffin and
slides processed for periodic acid-Schiff (PAS) reaction. For each sample, villus
TRIAL II
height, crypt depth and intraepithelial lymphocytes (IEL) were measured. The Goblet
cells in the villi and crypts were also counted. All measurements were made in 10
well-oriented villi and crypts.
8.2.4. Statistical Analysis
TRIAL III
The effect of diet on different parameters was tested with an ANOVA using the
GLM procedures of SAS statistics package (SAS Institute, INC. 8.1, Cary, NC). For
performance analyses, pig was used as the experimental unit for ADG, and pen for
ADFI and feed efficiency. Initial live weight was used as a covariate for productive
performance results. In slaughter measurements, the pig was the experimental unit.
TRIAL IV
In the event that significant diet effects were established (P < 0.05), multiple
comparisons of the means were performed using the PDIFF function of SAS adjusted
by Tukey Kramer. Faecal consistency data were analyzed by a χ2 test using the same
statistical software. Statistical significance was accepted at P < 0.05.
TRIAL V
135
INTRODUCTION
Trial V
Starter
Corn flakes
360.6
180.0
Barley
-
215.7
Wheat
-
100.0
Wheat flakes
240.2
120.0
Soybean meal, 44% CP
-
93.0
Full fat extruded soybeans
40.0
93.0
Soya protein concentratea
60.0
60.0
Wheat gluten
60.0
-
Potato protein
30.0
-
Fat-filled sweet whey
30.0
30.0
Sweet whey
150.0
80.0
Calcium carbonate
-
7.0
Calcium phosphate (dicalcium) (18%)
16.0
9.0
Salt
-
2.0
L-Lysine HCl 99%
5.0
3.0
DL-Methionine 99%
0.3
0.9
L-Threonine 98%
0.7
0.7
L-Tryptophan 10%
0.2
-
Choline HCl 50%
2.0
0.7
Vitamin and mineral pre-mixb and additives.
5.0
5.0
a
Soya HP-300. Hamlet protein A/S (Spain).
b
Provided the following per kilogram of diet: vitamin A, 10000 IU; vitamin D3, 2000 IU;
OBJECTIVES
Pre-starter
TRIAL I
Ingredient, g/kg
TRIAL II
Experimental period
TRIAL III
Phase 1 and Phase 2.
LITERATURE
REVIEW
Table 8.1. Composition as fed basis of pre-starter and starter control diets of
vitamin E, 15 mg; vitamin B1, 1.3 mg; vitamin B2, 3.5 mg; vitamin B12, 0.025; vitamin
B6, 1.5 mg; calcium pantothenate, 10 mg; nicotinic acid, 15 mg; biotin, 0.1 mg; folic
136
TRIAL V
mg; I, 0.75 mg; Se, 0.10; etoxiquin, 0.15 mg.
TRIAL IV
acid, 0.6 mg; vitamin K3, 2 mg; Fe, 80 mg; Cu, 6 mg; Co, 0.75 mg; Zn, 185 mg; Mn, 60
INTRODUCTION
Chapter 8
8.3.Results
LITERATURE
REVIEW
8.3.1. Growth performance
Results for body weight (BW), average daily gain (ADG), average daily feed
intake (ADFI), and feed efficiency (G:F) are shown in Table 8.2. Body weight or
ADG of the animals did not show significant differences between treatments. Intakes
were similar between treatments showing a progressive increase from 155 g/d during
OBJECTIVES
the first week to 825 g/d during the fifth week. For the first day post-weaning, intake
was very low and animals did not eat more than 100 g/d. During the second day there
were a compensatory response with a sudden increase in intake up to 170 g/d that
normalised thereafter (Figure 8.1). Feed efficiency was increased with the use of the
different additives compared to the control diet during the starter period (P < 0.05).
Regarding mean values for the whole experimental period, both additives and their
TRIAL I
combination improved efficiency ratio compared to controls (P < 0.05), with BP’ and
BMP treatment animals demonstrating higher values than BM (P < 0.05).
Figure 8.1. Voluntary feed intake (kg/day) of pigs receiving a control diet (CT),
TRIAL II
or the same diet supplemented with Bio-Mos (BM), Bioplex-Zn (BP’) or both
additives (BMP) during the first week post-weaning.
CT
BM
BP
BMP
TRIAL III
TRIAL IV
Voluntary feed intake, kg/day
0,30
0,25
0,20
0,15
0,10
0,05
0,00
d1
d2
d3
d4
Days
TRIAL V
137
d5
d6
d7
Table 8.2. Initial and final pig body weight (kg), voluntary feed intake (kg/day),
INTRODUCTION
Trial V
Dietsa
CT
BM
BP’
BMP
SEM
Diet pvalue
Initial body weight
6.65
6.64
6.63
6.68
0.207
0.99
Finalbody weight
17.81
17.77
18.09
18.73
0.782
0.82
Pre-starterb
0.24
0.22
0.24
0.24
0.011
0.58
Starterc
0.68
0.64
0.66
0.69
0.042
0.82
Whole period
0.50
0.47
0.49
0.51
0.023
0.54
Pre-starter
0.15
0.14
0.15
0.16
0.032
0.77
Starter
0.43
0.44
0.44
0.47
0.039
0.72
Whole period
0.35
0.34
0.35
0.37
0.019
0.72
0.57
0.58
0.66
0.66
0.033
0.15
z
x
x
x
Item
LITERATURE
REVIEW
average daily gain (kg /day) and feed efficiency in early-weaned pigs
feed
intake,
Average Daily Gain, kg/day
TRIAL I
Voluntary
kg/day
OBJECTIVES
Pig body weight, kg
Pre-starter
Starter
0.63
Whole period
0.63z
0.69
0.67
0.68
0.014
0.04
0.64y
0.66x
0.67x
0.009
0.002
TRIAL II
Feed efficiency
a
b
Period between the 1 to 2nd week post weaning.
c
Period between the 3 to 5th week post weaning.
d
Period between the 1 to 5th week post weaning.
x, y ,z
Least-squares means within a row lacking a common superscript letter differ (P <
0.05).
TRIAL III
Diets: CT (control diet), BM (control diet with 0.2 % Bio-Mos), BP’ (control diet with
0.08 % Bioplex Zn) and BMP (control diet with 0.2 % Bio-Mos and 0.08 % Bioplex Zn).
Faecal consistency was scored during the first three weeks post-weaning.
Percentage of pens with score two and three is shown in Figure 8.2. There was an
increase in faecal inconsistency at day four for all of the diets that tended to be higher
TRIAL IV
8.3.2. Faecal consistency
for CT diet (diet overall effect, P = 0.11) with more than 80 % of the pens with faecal
138
TRIAL V
inconsistency. During the following days, BM showed the fastest recovery with no
INTRODUCTION
Chapter 8
animal with faecal inconsistence at the end of the first week while the rest of diets
showed 25, 75, and 75 % for BP’, BMP and CT respectively (diet overall effect P <
LITERATURE
REVIEW
0.001). The controlled stress performed on the 14th day of the experimental period,
was reflected by an increase in faecal inconsistency on day 15 that was similar across
all dietary treatments.
Figure 8.2. Faecal consistency (% of boxes with a score value 2 and 3) in pigs
OBJECTIVES
receiving a control diet (CT), or the same diet supplemented with Bio-Mos (BM),
Bioplex-Zn (BP’) or both additives (BMP) during the initial three weeks postweaning
TRIAL I
TRIAL II
Faecal inconsistency, percentage of boxes
CT
BM
BP
BMP
100%
80%
60%
40%
20%
0%
d1
d2
d3
d4
d5
d6
d7
d8
d9 d10 d11 d12 d13 d14 d15 d16 d17 d18 d19 d20 d21
Days
8.3.3. Organ weights and small intestine length
TRIAL III
Weights and lengths from different compartments of the gastrointestinal tract at
the end of starter period are shown in Table 8.3. In general, the experimental diets did
not promote changes in weights or lengths of the different sections studied or their
contents. Only the empty ileal weight showed changes related to the diets with BP’
treatment having the highest values (8.9, 9.6, 11.9 and 10.3 for CT, BM, BP’ and
TRIAL IV
BMP respectively; P = 0.08).
8.3.4. Short Chain Fatty Acids (SCFA)
Total SCFA, pH and lactic acid concentrations in the stomach, ileum and caecum
are shown in Table 8.4. The pH was not modified by the experimental diets in any of
TRIAL V
139
the sections. As expected, fermentation products increased slightly from the stomach
INTRODUCTION
Trial V
either in total SCFA concentration or in their components (acetate, propionate,
butyrate, valerate and branched chain fatty acids, data not shown) in any of the
sections. Lactate concentration was also similar between diets.
LITERATURE
REVIEW
to the ileum and abruptly to the caecum. No differences were found between diets
gastrointestinal tract from early-weaned pigs sacrificed two weeks post-weaning
Dietsa
Item
CT
BM
BP’
BMP
SEM
Diet pvalue
OBJECTIVES
Table 8.3. Weight (g/kg BW) and length (m) of different parts of the
150.5
146.9
150.7
162.2
6.64
0.41
Stomach
Full
30.70
26.85
27.81
31.19
2.66
0.59
Empty
7.76
7.31
7.43
7.95
0.27
0.35
Content
22.95
19.55
20.37
23.25
2.56
0.76
Full
84.32
84.04
85.76
84.97
3.88
0.90
Empty
53.38
55.27
58.47
58.05
2.71
0.51
Content
30.95
26.77
27.30
26.27
1.99
0.31
Empty ileum
8.91
9.56
11.92
10.34
0.82
0.08
Full
35.43
38.03
37.11
48.81
4.15
0.12
Ileum
1.55
1.55
2.00
1.63
0.178
0.92
Whole
10.60
10.23
11.07
9.74
0.324
0.66
Ratio
0.15
0.15
0.19
0.17
0.016
0.82
Small
intestine
Large
intestine
Length, m
Small
intestine
0.08 % Bioplex Zn) and BMP (control diet with 0.2 % Bio-Mos and 0.08 % Bioplex Zn).
x, y ,z
Least-squares means within a row lacking a common superscript letter differ (P <
0.05)
140
TRIAL IV
Diets: CT (control diet), BM (control diet with 0.2 % Bio-Mos), BP’ (control diet with
TRIAL V
a
TRIAL II
Full
TRIAL III
Whole
intestine
TRIAL I
Weight, g
INTRODUCTION
Chapter 8
Table 8.4. pH, SCFA and lactic acid concentration (µmol/g dry matter (DM)) in
the stomach, ileum and caecum in pigs sacrificed two weeks post-weaning
LITERATURE
REVIEW
Dietsa
pH
OBJECTIVES
TRIAL I
CT
BM
BP’
BMP
SEM
Diet pvalue
Stomach
3.2
3.09
3.25
3.25
0.293
0.98
Ileum
6.82
6.7
6.74
6.69
0.107
0.81
Caecum
5.73
5.59
5.66
5.68
0.135
0.91
Stomach
8.31
7.48
6.24
9.6
1.048
0.17
Ileum
14.56
12.93
9.84
12.48
2.009
0.43
Caecum
130.1
136.3
132.8
137.8
7.478
0.89
3.68
3.75
4.80
0.594
0.51
Item
Total
SCFA
Lactic
Acid
a
4.35
Stomach
Ileum
15.84
9.85
14.34
14.20
3.845
0.72
Caecum
3.29
2.25
1.40
2.10
1.648
0.87
Diets: CT (control diet), BM (control diet with 0.2 % Bio-Mos), BP (control diet with
0.08 % Bioplex Zn) and BMP (control diet with 0.2 % Bio-Mos and 0.08 % Bioplex Zn).
TRIAL II
8.3.5. Quantitative changes in microbial population
Table 8.5 shows the concentration of purine bases in the ileum, caecum and
rectum digesta as an estimate of the total microbial population size and activity.
TRIAL III
Population size of lactobacilli and enterobacteria in the distal jejunum are also shown.
Purine base concentration increased drastically from the ileum to the caecum, without
differences between diets (6.46, 47.65 and 35.04 µmol/g DM for ileum, caecum and
rectum respectively). However it is interesting to remark that in both the caecum and
rectum, BM promoted the lowest mean values.
TRIAL IV
Lactobacilli did not show differences between diets, however, enterobacteria
showed a significant decrease in their numbers with BM and BMP treatment.
Compared to control (CT), enterobacteria decreased from 9.13 to 8.05 gene copies /g
FM with BM (P = 0.05) and from 9.13 to 7.89 with BMP (P < 0.05). Expressed as
TRIAL V
141
lactobacilli:enterobacteria, the BMP diet promoted the highest ratio compared to CT
INTRODUCTION
Trial V
8.3.6. Immune proteins and intestinal morphology
Table 8.6 shows serum concentrations of IgG, IgM and IgA, ileal concentration of
LITERATURE
REVIEW
(P = 0.03).
IgA and histological measurements performed in the distal jejunum wall samples. No
immunoglobulins, ileal IgA or in the number of intraepithelial lymphocytes or goblet
cells in the jejunum. However, crypt depth showed lower mean values with
experimental diets compared to CT, although differences only reached statistical
OBJECTIVES
significant differences among diets were found in the serum concentration of
difference for the BMP diet (P = 0.04). The villus height was not affected and this
resulted in a response in villus:crypt ratio similar to crypt depth, with significant
TRIAL I
increases when BM and organic Zn were both added into the diets.
8.4. Discussion
Numerous studies have reported that BM supplementation during the postDavis et al., 2002, 2004a). Recently, a meta-analysis of 54 different experiments in
nursery pigs fed with Bio-Mos demonstrated a 4.12 % improvement in weight gain,
2.11 % improvement in feed intake and an increase in feed efficiency of 2.29 %
TRIAL II
weaning phase improves growth performance of pigs (Dvorak and Jacques, 1998;
(Miguel et al. 2004). In the case of organic sources of Zn, results are scarce and more
controversial. Authors such as Carlson et al., (2004) did not demonstrate
organic zinc (Zn-polysaccharide or Zn-chelate) however, other researchers suggest
that Zn chelate (Bioplex Zn) may improve growth performance in young pigs (Mullan
TRIAL III
improvements in growth performance when nursery pigs were fed different sources of
et al., 2002; 2004, Case and Carlson, 2002). In our case, the inclusion of either BM
or BP
promoted an increase in feed efficiency compared to controls, although
significance. Growth promoting effects of feed additives are normally maximised
when animals are reared under field conditions where disease challenges are greater
than in an experimental farm situations where hygiene and environment are carefully
TRIAL IV
differences promoted in ADG or DFI were not large enough to reach statistical
controlled (Spring, 2004). Considering this, our improvements registered in feed
142
TRIAL V
conversion can be considered as a promising result of the potential of both additives
INTRODUCTION
Chapter 8
under practical conditions. In relation to the possible benefits of using BM and BP
together, from our study we could not detect any significant improvement in growth
LITERATURE
REVIEW
performance compared with the additives alone. Le Mieux et al. (2003) in a series of
four experiments evaluated BM and ZnO supplementation in nursery pigs with
variable responses. In general, the response of growth performance to BM was more
consistent with low levels of Zn, although in some of the trials BM addition was
effective in the presence of an excess of Zn which was manifest as an improvement in
gain:feed ratio. Similar variable results were described by Davis et al. (2004b) with a
OBJECTIVES
major response of BM when diets did not include an excess of Zn.
8.4.1. Changes on microbial ecosystem
BM has been proposed to promote growth by modifying the gastrointestinal
ecosystem, and reducing intestinal pathogen colonisation. This ability seems to be
TRIAL I
due to the capacity of BM to attach to mucosa binding proteins on the cell surface of
some bacteria, preventing colonisation of intestinal epithelium (Spring et al., 2000).
In that sense, we found a selective diminution in the enterobacteria population in pigs
fed diets supplemented with BM (BM and BMP diets), but no change was registered
with Zn addition. Likewise, White et al. (2002) found a lower concentration of
TRIAL II
coliforms in the faeces of pigs fed diets with MOS. The decline in enterobacteria
numbers is noteworthy because of the relationship that this group of bacteria have
with post-weaning diarrhea syndrome. The ratio of lactobacilli:enterobacteria was
first proposed by Muralidhara et al. (1977) as an index of robustness of commensal
microbiota. An inhibition of these bacteria could prevent or decrease the severity of
TRIAL III
diarrhea that appears during the initial days after weaning (Gianella, 1983). This
could be the reason why animals receiving the BM diet showed a more rapid recovery
after the outbreak of faecal inconsistency observed at four days post-weaning.
TRIAL IV
TRIAL V
143
Table 8.5. Purine Bases concentration (µmol/g DM) in the ileum, caecum and rectum, and bacterial populations (Lactobacilli and
enterobacteria) from the distal jejunum measured by Real-Time PCR (log 16S rDNA gene copies /g FM) in ileum digesta in early-weaned
pigs
Item
Dietsa
Purine Bases
Bacteria
a
CT
BM
BP
BMP
SEM
Diet p-value
Ileum
6.17
5.62
6.58
7.46
1.38
0.81
Caecum
50.23
40.38
52.86
47.16
5.64
0.45
Rectum
41.28
25.28
34.79
40.00
6.25
0.19
10.04
9.62
9.76
9.79
0.27
0.75
x
y
8.87
7.89
y
0.28
0.01
0.89y
1.89x
0.81
0.04
Jejunum
Lactobacilli
Enterobacteria
9.13
8.05
Lactobacilli : enterobacteria
0.91y
1.57xy
xy
Diets: CT (control diet), BM (control diet with 0.2 % Bio-Mos), BP (control diet with 0.08 % Bioplex Zn) and BMP (control diet with 0.2 % Bio-
Mos and 0.08 % Bioplex Zn).
TRIA
L II
TRIA
L III
TRIAL V
Least-squares means within a row lacking a common superscript letter differ (P < 0.05).
TRIA
L IV
x, y ,z
TRIAL V
Table 8.6. Plasma and ileal immunoglobulin (IgA, Ig M and IgG) concentration and jejunum histological parameters in early-weaned
pigs sacrificed two weeks post-weaning
Dietsa
Item
CT
BM
BP
BMP
SEM
Diet P-value
Ig G, mg/ml
25.50
20.97
25.10
13.88
4.142
0.26
Ig M, mg/ml
4.40
5.44
6.81
5.05
0.853
0.32
Ig A, mg/ml
0.82
1.43
1.48
1.05
0.255
0.34
IgA/gDM
1.47
2.01
1.72
1.94
0.470
0.85
IgA/Gcp
6.19
8.29
7.04
7.42
1.670
0.84
Crypt depth, μm
281.3x
241.60xy
240.10xy
234.50y
11.980
0.04
Villus height, μm
338.5
340.80
328.50
335.10
19.420
0.97
Villus: crypt ratio
1.23y
1.41xy
1.40xy
1.46x
0.060
0.05
Lymphocytes/100 enterocytes
4.64
3.60
4.66
5.16
0.940
0.69
Crypt Goblet cells/100 enterocytes
17.56
15.33
15.70
15.51
1.071
0.44
Villus Goblet cells/100 enterocytes
3.36
3.35
3.83
2.91
0.604
0.76
Inmunoglobulin
Plasma
Ileum digesta
TRIA
L II
a
TRIA
L IV
Jejunum wall
TRIA
L III
Morphology
Diets: CT (control diet), BM (control diet with 0.2 % Bio-Mos), BP (control diet with 0.08 % Bioplex Zn) and BMP (control diet with 0.2 % Bio-
Mos and 0.08 % Bioplex Zn).
Changes in bacterial populations were not reflected in fermentation patterns that
INTRODUCTION
Trial V
inability of feed added mannan oligosaccharides to modify faecal pH or VFA
concentration in weaning pigs (White et al., 2002) and neither in dogs (Swanson et
al., 2002). Although non-digestible fermentable oligosaccharides such as fructo
oligosaccharides (FOS) are considered as VFA-promoting compounds due to their
LITERATURE
REVIEW
were unaffected by the experimental diets. Similarly other authors have related the
high susceptibility to be fermented preferentially by some lactobacilli and
compared to other non-digested carbohydrates probably preclude their potential to
promote different fermentation patterns. From our results it seems more plausible
that the specific effect of BM on enterobacteria populations is related to an
OBJECTIVES
bifidobacteria species (Kaplan and Hutkins, 2000), their low inclusion rate in the diet
impairment of gut colonisation by these bacteria, presumably by a specific blockage
One of the modes of action proposed for the use of pharmacological levels of
ZnO as a growth promotant has been their potential antimicrobial properties
(Sordeberg et al., 1990). Katouli et al. (1999) showed that high doses of Zn oxide
TRIAL I
of their binding sites as has previously proposed (Spring et al., 2000).
increased the stability of the intestinal microflora through a reduction in the diversity
of coliform species that the authors regarded as an index of a more robust microbiota.
absence of quantitative effects on coliform populations was also described by JensenWaern et al. (1998) using 2500 ppm of ZnO in weaners with no effects on coli and
TRIAL II
However, no significant effect was found in the total number of coliforms. The
enterococci in faeces. This lack of a direct effect on specific bacterial populations
with pharmacological levels of ZnO make it difficult to consider that organic sources
account that they are added to a much lower doses.
TRIAL III
of Zn play their role through a direct effect on intestinal microbiota when taking into
8.4.2. Effect on gut function
The early-weaned piglet has to cope with a massive challenge with antigens
intestinal flora. Facultative pathogens, previously controlled by maternal IgA can
expand and colonise the gut. The maintenance of the intestinal integrity and the
TRIAL IV
associated with the new food and with the establishment of a novel commensal
digestive and absorptive function during this weak period depends on the ability of
146
TRIAL V
the immune system to adapt to this new situation and to discriminate between
INTRODUCTION
Chapter 8
“harmful” and “harmless” antigens with an appropriate response (Bailey et al., 2001).
External aggressions to the enterocyte, as those caused by microorganisms or new
LITERATURE
REVIEW
feed proteins are normally associated with an atrophy of intestinal villi compensated
at least partially by an accelerated turnover of crypt cells which results in a reduced
villi:crypt ratio (Miller et al., 1986). In this experiment, the addition of BM or BP to
the diets promoted a decrease in the mean values for crypt depth, although differences
with control only reached statistical significance when both additives were combined
into the BMP diet. A similar response was registered in the villi:crypt ratio as villus
OBJECTIVES
height remains unaffected by the experimental treatments. Other authors have
previously described the effect of BM addition on gut structure. Similar to the results
reported here, Ferket (2002) found that adding 0.1 % Bio-Mos to broilers did not
affect villus height, but promoted a decrease in crypt depth with a lower villi:crypt
ratio. Other authors such as Iji et al. (2001) also observed an increase in such a ratio
in poultry, but due to a significant increase in villi height rather than crypt depth.
TRIAL I
This beneficial effect of BM on intestinal morphology may respond to the observed
reduction in the enterobacteria population, but may also be due to other mechanisms.
Ferket et al. (2002) have also proposed an increase in the production of the mucous
gel layer promoted by BM as another mode of action of mannan-oligosaccharides.
The mucous layer acts as barrier against bacterial aggression thus protecting the host
TRIAL II
animal from enteric infection. The current experiment did not detect any increase in
the number of Goblet cells responsible for the production of mucous on villi or
crypts. Stimulating effects on mucin dynamics in the presents of BM could be
attributed to an increase in mucin gene expression rather than the increase in actual
goblet cells (Smirnov et al 2005).
TRIAL III
The use of BM as a tool to modulate immune response has been demonstrated by
(O'Quinn et al. 2001) who found increased IgA titers in sow’s milk or Davis et al.
(2004a) who reported an alteration in the leukocytes populations in piglets fed BM.
Preventing the onset of an acute phase immune response by modulating the immune
response has a profound impact on growth performance.
None of the inmunological
TRIAL IV
parameters included in this study, like plasmatic and intestinal Igs or intraepithelial
lymphocytes, responded significantly to the inclusion of BM, however it is possible
that these broad indexes are not sensitive enough to detect more subtle effects on
immune response.
TRIAL V
147
The mode of action of supplemental Zn as a growth promotant in young pigs
INTRODUCTION
Trial V
systemic effect. High feed concentrations of Zn (2000-3000 ppm of ZnO) has become
a common practice to control post-weaning diarrhea with probed positive results, but
it has raised some environmental concerns because of the high release of Zn into the
environment. If Zn promoted growth throughout a systemic effect, the use of organic
LITERATURE
REVIEW
remains unclear, and there are doubts if it is based on a luminal, an intestinal or a
sources of Zn with higher bioavailability could represent a good alternative. Zinc is
also functions as an antioxidant protecting cells from the damaging effects of oxygen
radicals generated during immune activation (Bray and Bettger, 1990). Protection
against deleterious effects of inappropriate immune responses against bacterial or new
OBJECTIVES
know to play a central role in the immune system (Shankar and Prasad, 1998), and
antigens after weaning could explain the increase that we observed in villus:crypt
ratio. In addition to an increased protection, an improvement of the development of
in the plasmatic immunoglobulins related to diets, however BP diet showed the
highest Ig M and Ig A plasmatic mean values, whereas the control diet showed the
TRIAL I
the immune response can also be suggested. We could not find significant differences
lowest. In addition, it is interesting to note that animals fed organic zinc showed a
heavier empty ileum, which was numerically longer compared with the rest of the
showing a continuous Peyer’s Patch. Therefore, a higher ileal weight may indicate a
higher development of the Peyer’s Patches in these animals. In the newborn piglet the
TRIAL II
treatments. In this work, the ileum was considered as the section of the small intestine
mucosal immune system is almost completely absent, and during the first weeks after
birth rudimentary Peyer’s Patch follicles expand rapidly and a spatially organised
Supplementing an additional organic source of Zn could aid the suitable development
of the immune response in the young pig. This could explain the positive result
observed both in intestinal architecture and in the development of the Peyer’s
TRIAL III
architecture of the mucosal immune system takes place (Bailey et al., 2001).
Patches.
The use of Bio-Mos® derived from the outer yeast cell wall of a selected strain
of Saccharomyces cerevisiae and Bioplex Zn™
supplement improved growth
performance in terms of feed efficiency in the weaning pig. The mode of action of
TRIAL IV
8.5. Implications
BM seems to be related to the inhibition of certain opportunistic gut bacteria from
148
TRIAL V
the Entobacteriaceae family, whereas organic Zn could act through an improvement
INTRODUCTION
Chapter 8
in the host immunological response, suggested by an increased ileal weight.
Complementary actions could explain the highest values in villus:crypt ratio when
LITERATURE
REVIEW
both additives were used together.
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
149
Chapter 9
DISCUSSION
LITERATURE
REVIEW
General discussion
150
TRIAL I
TRIAL V
TRIAL IV
TRIAL III
TRIAL I. Quantification of total bacteria, enterobacteria and lactobacilli
populations in pig digesta by real time PCR.
TRIAL II. Influence of weaning on caecal microbiota of pigs: use of realtime PCR and t-RFLP.
TRIAL III. Molecular analysis of bacterial communities along the pig
gastrointestinal tract.
TRIAL IV. The response of gastrointestinal microbiota to the use of
avilamycin, butyrate and plant extracts in early weaned pigs.
TRIAL V. Use of mannan-oligosaccharides and zinc chelate as growth
promoters and diarrhea preventative in weaning pigs: effects on microbiota
and gut function.
TRIAL II
The following chapter discusses the overall results obtained in the different
experiments included in this thesis (Chapter 4-8):
OBJECTIVES
GENERAL DISCUSSION
DISCUSSION
LITERATURE
REVIEW
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
9.1. Usefulness of quantitative PCR, FISH and t-RFLP to study the intestinal
microbiota
In the last fifteen years, molecular methodology has been increasingly used in
gastrointestinal
microbiology,
greatly
improving
the
knowledge
regarding
DISCUSSION
LITERATURE
REVIEW
General discussion
Although traditional methods have greatly contributed to set up the basis of
gastrointestinal microbiology, it is well known that these methods are inherently
limited by their low sensitivity, reproducibility, labourity and inadequacy in detecting
all gut bacteria (Furham et al., 1992; Dutta et al., 2001). Recent works estimate that
OBJECTIVES
composition, phylogeny and function of this complex ecosystem.
only a range of around 10-40% of the gastrointestinal microbiota can be accounted
have several advantages compared with traditional ones: the viability of the cells is
not required, thus avoiding the need to work in fresh and also the ability of bacterial
cells to growth in a medium semisynthetic that appear as a critical point for most of
the traditional ones. Moreover, once implemented they are less cumbersome than
traditional methods, with high levels of sensitivity and reproducibility (Wang et al.,
1996, Raskin et al., 1999; McCartney, 2002). However, when required, isolation of
DNA from the digesta can turn on a limitation step that may involve differences in
DNA isolation efficiency of different bacteria (McOrist et al., 2002; Anderson and
Lebepe-Mazur, 2003), DNA extraction being a source of bias in the representativity
of the whole ecosystem. In this regard, a consensus between different working groups
in the method used to extract DNA might be extremely useful to obtain results more
comparable than at present, avoiding this bias. Also, it is necessary to remark that
many of these molecular methods used for characterization of species composition of
the microbiota (known as fingerprinting methods) could also be biased on the first
PCR amplification of the 16S rRNA gene using universal primers. The proper
election of primers (Liu et al., 1997; Osborn et al., 2000), number of PCR cycles
(Suzuki and Giovanonni, 1996) and PCR conditions (Kitts, 2001) is essential to
achieve a direct proportion between the abundance of amplicons to the abundance of
that template in the sample (Clement et al., 2000; Dunbar et al., 2000).
152
TRIAL I
for by traditional methods (Zoetendal et al., 1998). In this regard, molecular methods
DISCUSSION
Chapter 9
In the different trials included in this thesis, three different molecular methods
were used: real-time PCR (qPCR), terminal-Restriction Fragment Length
LITERATURE
REVIEW
Polymorfism (t-RFLP) and Fluorescent in situ hybridization (FISH).
To quantify total and particular bacterial groups, two different methods were
used: qPCR (trial I, II, IV and V) and FISH (trial III), the first one as a rapid
method to assess robustness of microbiota measured as lactobacilli:enterobacteria
ratio and the second one to obtain an overall picture of the main bacterial groups
OBJECTIVES
described in the pig.
Real-time PCR with Sybr Green® dye was employed to quantify total bacteria,
lactobacilli and enterobacteria, as objective bacterial groups in pig microbiology.
Recently, other research groups have also described quantification of total bacteria
and lactobacilli in pig gut digesta by real-time PCR (Collier et al., 2003; Hill et al.,
2005). First results obtained (trial I) were compared with those obtained by
TRIAL I
traditional methods (culture and direct microscopy) to assess its usefulness.
In our case, the results obtained by qPCR were higher than those with traditional
methods, although similar discrepancies have often been described (Nadkarni et al.,
2002; Bach et al., 2002; Huijsdens et al.,2002). Different facts may be behind
TRIAL II
and free DNA present in the samples and thus amplified by real-time PCR but not
quantified by traditional methods (Rigottier-Gois et al., 2003). Secondly, the
multiplicity of 16S rRNA gene copies (7 for E. coli and 4 for Lactobacillus spp.;
Fogel et al., 1999). And thirdly, differences intrinsic to methods, in particular the pretreatment of digesta in direct microscopy and culture that may involve a loss of an
TRIAL III
important fraction of the bacteria firmly attached to particulated material when
previous dilution is done. In this regard, we were able to detect losses of microbial
material up to 90%. Finally, quantification of non-specific amplicons with Sybr
Green dye have also been described (Hein et al., 2001), although in our case, melting
curve analysis was performed and discarded this possibility (Figure 9.1).
TRIAL IV
Despite differences in absolute numbers, correlation in total bacteria and in the
lactobacilli: enterobacteria ratio between traditional and molecular method used,
confirmed validation of the results obtained. For this reason, the method was
considered as a useful technique to assess changes in microbial ecosystem rapidly by
TRIAL V
153
TRIAL II
differences observed. Firstly, presence of non- viable, viable but not culturable cells
DISCUSSION
General discussion
the use of the ratio lactobacilli:enterobacteria, avoiding comparison between absolute
Figure 9.1.
Melting curve obtained after the PCR reaction for total (A),
enterobacteria (B), and lactobacilli (C). Dissociation temperature (ºC) for PCR
LITERATURE
REVIEW
values.
B
C
TRIAL I
A
OBJECTIVES
product plotted vs. the signal fluorescence derivative.
This ratio, as previously mentioned, has been routinely used as an indicator of gut
health (Muralidhara et al., 1977; Ewing and Cole, 1994) with an increase in the ratio
being considered beneficial for the animal gut health. Interest in both these bacteria
becomes from the fact that whereas lactobacilli have been associated with favorable
effects on animal health, bacteria belonging to the Enterobacteriaceae family are
related with diarrhea outbreaks (Melin, 2001). Lactobacilli is thought to promote
health through inhibition of some opportunistic pathogens, such as E. coli (Blomberg
et al., 1993; Tannock et al, 1999), by preventing or decreasing the severity of diarrhea
that appears during the initial days after weaning (Gianella, 1983) and also by
modulating an adequate immune response (Perdigón et al., 2001). Due to this, the
objective is to maintain a ratio favorable to lactobacilli, especially in young pigs.
However, it is fair to remark that literature on the ratio is scarce. Results come
from previous works that described changes by traditional methods in intestinal
bacteria in pigs of different ages and experimental conditions (Chopra et al., 1963,
Muralidhara et al., 1977; Reid and Hillman, 1999; Manzanilla et al., 2004). In this
154
DISCUSSION
Chapter 9
regard, further investigations are needed to verify the usefulness of this ratio to check
pig gut health and also to see if it could have some kind of relationship with
LITERATURE
REVIEW
performance improvement.
Table 9.1. Results of lactobacilli:enterobacteria ratio and its relation with growth
performance from pigs included in trial II, IV and V.
II
Item
Ratio
lactobacilli:enterobacteria
Improvement in growth
performancea
Section
/Performance
measure
Diet
S
W
Caecum
-0.27y
-1.76x
Average daily
gain
+
TRIAL I
CT
IV
Ratio
lactobacilli:enterobacteria
Improvement in growth
performance
AB
Jejunum
3.30
2.02
Caecum
z
z
0.48
0.43
AC
XT
2.32
2.23
0.75
yz
1.10y
TRIAL II
Average daily
gain
t
t
t
Feed
efficiency
+
+
=
BM
BP’
BMP
CT
Ratio
1.57xy
0.89y
1.89x
Jejunum
0.91y
lactobacilli:enterobacteria
V
Improvement in growth
Feed
+
+
+
performance
efficiency
a
Overall results observed in growth performance (-, indicates significant impairment, +,
= not significant changes and t, a tendency to improve).
TRIAL III
In our case, the ratio was determined in trials II, IV and V (Table 9.1), where the
piglets were always healthy. In general, when the index increased the animals always
showed an improvement in performance, compared to control treatment. In particular,
in trial II, comparing suckling with weaned pigs, those that remain with the mother
TRIAL IV
show the high ratio parallel to higher lower body weight. Similarly, in trial IV,
animals that received the plant extract showed the highest lactobacilli:enterobacteria
ratio in cecum with an improvement in average daily gain. In trial V, the ratio was
significantly increased in the jejunum digesta of animals that received both additives
together (mannan-oligosaccharides plus organic zinc) with an increase also in the
TRIAL V
155
TRIAL II
OBJECTIVES
Trial
DISCUSSION
General discussion
feed:gain ratio. However, when the performance improved we could not always see
performance in addition to those related directly with microbiota, that may behind the
results obtained.
Besides qPCR, FISH was also used to quantify total bacteria, but in this case the
LITERATURE
REVIEW
an increase in the ratio. Undoubtly, several factors are implied in improving pig
objective was to cover most of the main microbial groups of the pig gastrointestinal
Bacterial groups determined were: Bacteroides/Prevotella group, Ruminococcus
flavefaciens, R. bromii, clostridia cluster XIVa, clostridia cluster IV species related to
Faecalibacterium prausnitzii, clostridia cluster IX, Streptococcus/Lactococcus sp.
and Lactobacillus/Enterococcus sp. (trial III). The aim of this trial was to study
OBJECTIVES
tract with the different probes used to give a overall picture of the ecosystem.
composition of adult pig gut microbiota along the gastrointestinal tract and also the
been extensively used to study other ecosystems (Amann et al., 1996; Hold et al.,
2003; Takada et al., 2004), its application in pig gut microbiology has been scarce; to
our knowledge, there are only two published works that have applied this method to
study pig gut microbiota: Konstantinov and co-workers (2004b), which used the
method to quantify total bacteria, Lactobacillus-enterococcus group, L. amylovorus
and L. reuteri-like in ileum and colonic digesta of weaning pigs; and recently,
Tzortzis and co-workers (2005) applied FISH to study the effect of the addition of a
novel galactooligosaccharide on Bacteroides spp, Bifidobacterium spp., Clostridium
hystoliticum group, and Lactobacillus/Enterococcus group of the gastrointestinal tract
of piglets.
In our case, the results obtained were more clarifying in distal sections of the
gastrointestinal tract due to the higher coverage obtained with the probes used. The
presence of bacteria belonging to different groups from those contained with the set
of probes used, may explain the lack of coverage in the upper gastrointestinal tract.
Of these groups, proteobacteria and also some other Clostridium groups that have
been described as important habitants of the pig gut (Leser et al., 2002) may be
behind differences obtained.
Regarding methodological aspects, a similar minimum level of detection was
obtained with FISH and qPCR (105-106 cells /g of digesta). However, FISH has a
main advantage compared to qPCR: DNA extraction and further DNA amplification
is not required, thus avoiding the possibility of the previously mentioned bias.
156
TRIAL I
potential of dietary fiber to manipulate its equilibrium. Although this method has
DISCUSSION
Chapter 9
Moreover, additional information regarding morphology of bacteria can be obtained
with FISH. The application of flow cytometry (Wallner et al., 1997; Moter and
LITERATURE
REVIEW
Göbel, 2000) to count hybridized bacterial cells would make this method highly
valuable in microbiological studies.
The bacterial profile based on polymorphism of the 16S rRNA gene was also
studied. The T-RFLP methodology was implemented and used in trial II, following
the method described by Höjberg and co-workers (2005). Results shown in trial III
OBJECTIVES
and IV included information obtained by RFLP method (Pérez de Rozas et al., 2003),
that is substantially similar to T-RFLP. This method has been used to study pig gut
microbiota by other research groups, to assess differences in microbial profiles after
dietary changes as fiber composition content of the diet (Leser et al., 2000; Högberg
et al., 2004), or the administration of different additives (Höjberg et al., 2005)
providing valuable information of changes in microbiota due to these dietary
TRIAL I
modifications.
In addition to bacterial profile obtained by t-RFLP, fingerprinting methods also
permit us to obtain the biodiversity of the samples (measured as number of bands)
which seems to be a useful index to assess gut microbiota stability and health. In this
TRIAL II
biodiversity, a higher number of species being an indicator of a more stable
ecosystem (Atlas, 1984) and thus a higher resistance against potential opportunistic
pathogens (Hillman et al., 2001). However, literature regarding pig gut biodiversity is
still scarce. In our case, results from trial IV showed an increase when different
additives were used, supporting thus the hypothesis that additives improved gut
TRIAL III
health. The animals had achieved a more diverse ecosystem and therefore more
difficult to be altered by opportunistic pathogens. Recently, other authors have been
observed changes in biodiversity of pig gut microbiota. Konstantinov and co-workers
(2003, 2004b) showed differences in biodiversity (measured as number of bands
obtained by DGGE) after addition of fermentable carbohydrates to the diet of
weaning pigs. The animals fed with fermentable carbohydrates in the diet showed a
TRIAL IV
higher biodiversity. Similarly, Högberg and co-workers (2004) also found differences
in pig microbial biodiversity obtained by t-RFLP in ileum of growing pigs after
feeding diets with different types and quantities of non-starch polysaccharides. In this
case, a lower biodiversity was obtained in animals fed insoluble non-starch
polysaccharides.
Recently, Inoue and co-workers (2005), using TGGE method
TRIAL V
157
TRIAL II
regard, the robustness of a microbial ecosystem has been directly related to its
DISCUSSION
General discussion
described the evolution of pig gut microbiota in the first weeksof life with a marked
On the other hand, the theoretical inference of bands obtained by restriction of
bacterial DNA in the samples with potential compatible species may be remarked.
Although it is need to keep in mind that results are dependent on sequences deposited
LITERATURE
REVIEW
increase in biodiversity after weaning.
in the database, interesting results have been obtained (trial II). Other authors have
to their potential compatibility using their own database obtained by cloning (Leser et
al., 2000). In our case, we inferred bacterial groups of those bands that appeared at
least in three animals, using public database software of RDP II (Cole et al., 2003). In
doing so, we covered around 30% of the total peak area, the remaining 70 % being
OBJECTIVES
also used similar procedures, but in this case only some particular bands were inferred
attributed to differences between animals, inherent background of the method, and
In conclusion, molecular methods used in this thesis can be considered useful new
tools for studying pig gut microbiota and for detecting changes in particular bacterial
groups. As with other techniques, there are limitations; however, new information
about the bacterial ecosystem structure is given by fingerprinting methods like tRFLP, and practical advantages like not needing to work in fresh, make these
methods especially attractive and complementary to traditional methodology and
commonly used in intestinal microbiologial studies.
9.2.Weaning: a critical stage in the indigenous pig microbiota establishment
As described in the previous chapters, pig gut colonization by bacteria is a
complex and successional process that takes several months to be completed (Swords
et al., 1993) and undergoes a marked disruption when piglets are separated from the
sow (Wallgren and Melin, 2001).
In nature, weaning is a transitional and long period of time in which mammals
change from total nutritional and social dependence on the mother to total
independence from her (Held and Mendl, 2001). However, the pig production system
involves very early and abrupt piglet weaning at a time when the immune system is
still immature (Bailey et al., 2001). As a consequence, animals refrain from eating,
with several negative consequences for their health that are reflected in the post158
TRIAL I
also to peaks without theoretical correspondance in the database used.
DISCUSSION
Chapter 9
weaning syndrome (Pluske et al., 1997; see Figure 2.2, chapter 2). In this regard, the
different trials completed with weaned pigs supported the stress described in pigs
LITERATURE
REVIEW
after weaning in different ways.
The shift in microbiota observed in trial II seems particularly remarkable. A
marked change in the bacteria inhabiting the piglet caecum was observed one week
after separation from the sow. This change was clearly reflected in the
lactobacilli:enterobacteria ratio, with a significant decrease in weaned pigs. In this
OBJECTIVES
regard, previous works have described increases in coliform bacteria in parallel with
decreases in lactobacilli after weaning (Jensen, 1998; Mathew et al., 1996; Franklin et
al., 2002).
In addition, the ratio was lower than that obtained in animals slightly older from
the rest of the trials (see Table 9.1). The short age of these piglets may explain the
high enterobacteria counts, due to the fact that those bacteria are one of the main
TRIAL I
groups that colonize piglet gut after birth, coming from the mother feces and the
environment (Swords et al., 1993; Ewing and Cole, 1994). The weaned pigs used in
the other trials (IV and V) were sacrificed at an older age than those in trial II, a fact
that may explain differences observed; those animals could have a more established
TRIAL II
et al., 2002).
The shift in microbiota profile was also demonstrated by t-RFLP profiles, which
clustered animals separately. As expected, weaned pigs showed a lower similarity
between them which again reflects the disbiosis suffered. The different compatible
bacteria inferred from the results obtained in this trial seem particularly interesting.
TRIAL III
Although theoretical, and therefore restricted, results can be considered as an image
of what is happening in this enormous ecosystem (Figure 9.2). Weaned pigs showed a
lower compatibility with lactic acid bacteria and also an absence of compatible bands
with bacteria such as C. coccoides and C. butyricum constantly present in suckling
pigs.
TRIAL IV
TRIAL V
159
TRIAL II
microbial profile with lactic acid bacteria as one of the main bacterial groups (Leser
DISCUSSION
General discussion
Figure 9.2. Pie chart with the major 5’-terminal fragments expressed as the mean
portion represents the mean of the percentage of total area compatible with a potential
bacteria.
SUCKLING
WEANED
Lactic acid
bacteria
L. acido phillus
Entero co ccus sp.
CFB phylum
L. delbruekii sp. Delbruekii
Clo stridium co cco ides
Escherichia
L. delbruekii sp. Lactis
Eubacterium, Rumino co ccus,
B utyrivibrio , Ro seburia, Clo stridium spp.
Fibro bacter succino genes
L. fructivo rans
Clo stridium spp.
Fibro bacter intestinalis
Lacto bacillus vaginalis
C. butyricum
Others
Although a lower biodiversity in coliform bacteria has usually been described at
weaning (Katouli et al., 1995; 1999), lactobacilli biodiversity have been less studied.
These changes undoubtedly reflect the new situation where the piglets are, with a
sudden change in the amount of type of substrate available for bacteria which results
in a transitional decrease in biodiversity. This fact could help to explain the lower
resistance of animals to potential pathogen colonization in the days following
weaning. It has been recognized that diverse bacterial population plays a key role in
the maintenance of the gastrointestinal health because it avoids potential colonization
by pathogens (Van Kessel et al., 2004). Therefore, avoiding a marked decrease of this
biodiversity can be especially important at weaning due to the fact that fluctuations
may be an excellent opportunity for opportunistic bacteria that contribute to digestive
disorders (Mathew et al., 1996). In this regard, the administration of probiotics,
160
TRIAL I
OBJECTIVES
Lactic acid
bacteria
LITERATURE
REVIEW
of the percentage of the total area in suckling (S) and weaned (W) group. Each
DISCUSSION
Chapter 9
mainly as lactic acid bacteria, might be a key strategy to avoid problems regularly
associated with commercial weaning.
LITERATURE
REVIEW
Another issue especially important in pig production, is the growth stasis
described after weaning (Le Dividich and Herpin, 1994; McCracken et al., 1995;
1999). In our case, trial II confirmed the results, performance being clearly affected
by weaning; weaned piglets showed a lower weight gain than their littermates that
remainded with their dams. Similarly, in trial V, where daily evolution of feed intake
OBJECTIVES
of weaned pigs was assessed during the first 7 days after weaning,
a marked drop
was found in the first 2-3 days, which was not recovered until 7th day post-weaning.
In this case, a peak of fecal inconsistence was detected on day 4; different causes
could be involved in this fact. Among these, low intake and changes in gut wall
arquitecture together with a microbiota unstabilization have been related with
diarrhea outbreaks after weaning (Pluske et al., 1997).
TRIAL I
9.2.1. Establishment of adult gut bacteria
TRIAL II
adult pig gastrointestinal tract was obtained. In agreement with literature, the total
bacterial load measured showed a clear increase from small intestine to rectum of
around 2 log units. Similarly to trial IV, with younger animals and qPCR 3 log units
of difference between stomach and distal colon were found. The dissimilar
environmental conditions mark the increase in population from proximal to distal
parts of the gut. Whereas peristaltic movement and acidic conditions in the upper tract
TRIAL III
impairs bacterial colonization (Ewing and Cole, 1994), the high quantity of substrate
and the lower rate passage improve colonization in caecum, colon and rectum
(Stewart et al., 1999). Other authors using FISH to count total bacterial load have
described similar values (Konstantinov et al., 2004b).
Moreover, gastrointestinal microbiota differs not only quantitatively, but also
TRIAL IV
qualitatively throughout the gut. In this regard, trial III shows us an interesting
description of main bacteria in the adult pig gut (stomach, jejunum, proximal colon
and rectum; Figure 9.3). As expected, different bacteria were found as main groups in
the upper and the lower gastrointestinal tract. However, the results obtained were
more clarifying in distal sections of the gastrointestinal tract, due to the higher
TRIAL V
161
TRIAL II
In trial III, an overall description of different bacteria inhabiting the growing-
DISCUSSION
General discussion
coverage obtained with the probes used. Results obtained confirm the dominance of
relatives in the large intestine, in comparison with stomach and jejunum where lactic
acid bacteria appeared as the predominant group. Results agree with literature where
lactic acid bacteria are described as the main bacteria in the upper gastrointestinal
tract (Reid and Hillman, 1999; Hill et al., 2005), and obligate anaerobes such as
LITERATURE
REVIEW
anaerobic bacteria related to clostridial clusters XIVa and to the clostridial cluster IV
eubacteria, clostridia and CFB phylum in the large intestine (Conway et al., 1994;
Figure 9.3. Total bacteria, Bacteroides/Prevotella group (probe Bac303),
clostridia cluster XIVa (Erec482), Faecalibacterium prausnitzii (Fprau645),
OBJECTIVES
Leser et al., 2002).
Ruminococcus flavefaciens and R. bromii (Rbro730 and Rfla729), clostridia cluster
IX
(Prop853),
Streptococcus/Lactococcus
sp.
(Str493)
and
Lactobacillus/
TOTAL
110
100
90of
80
70
60
50
40
30
20
10
0
BAC303
EREC482
FPRAU645
RBRO/RFLA
PROP853
TRIAL I
Enterococcus sp. (Lab158) measured by FISH in gastrointestinal tract.
STR493
LAB158
growing pigs (Trial III).
STOMACH
JEJUNUM
COLON
RECTUM
9.3. Are antibiotic-growth promoters a model to copy?
9.3.1. Mode of action of antibiotics: quantitative or qualitative effects on gut
microbiota?
Until their total ban in January 2006, antibiotics as growth promoters were
regularly used to improve feed utilization, growth, and to maintain piglet gut health.
Although they were used widely in recent decades, their exact mechanism of action is
162
DISCUSSION
Chapter 9
not completely known. The reduction of bacterial load in the upper gastrointestinal
tract, and therefore in the energy potentially available for the host but consumed by
LITERATURE
REVIEW
normal microbiota, is one of the main hypothesis postulated (Anderson et al., 1999;
Hardy et al., 2002).
However, results obtained in trial IV did not agree with this hypothesis, since the
antibiotic did not reduce total bacteria either in the upper or in the lower
gastrointestinal tract. Other authors have seen similar results when testing antibiotics.
OBJECTIVES
Collier and co-workers (2003) found a decrease in total bacteria on day 21 after
feeding pigs with tylosin that was recovered one week after, probably due to a
replacement of bacteria affected by other resistant strains. Moreover, an effect on
lactobacilli was expected, due to the spectra of avilamycin against gram positive
bacteria, however, this was not detected. In this regard, similar results have been
found before with absence of effect of avilamycin on lactobacilli counts (Decuypere
TRIAL I
et al., 2002).
Despite the lack of effect of avilamycin on total bacterial load, the dendogram
obtained by RFLP showed a clear separation of diets, a fact that could be behind a
marked change in species bacteria composition with the antibiotic used. This fact
TRIAL II
health of the animals not by reducing total bacteria load, but by changing species
composition becoming microbiota in a more favorable equilibrium for the host.
These results would suggest that the modulation of bacterial microbiota to achieve
an optimal equilibrium would be the strategy to substitute antibiotics as a growth
promotants instead of reducing total bacterial load as have been routinely proposed.
TRIAL III
9.3.2. Other in feed-additives with antimicrobial properties
In response to the need for alternatives to in-feed antibiotics, research and
development effort is being focused on the search for effective replacements. The
TRIAL IV
different trials included in this thesis aimed to evaluate some of the additives used
today in pig production (acidifiers, plant extracts, prebiotics and organic minerals),
with special interest in their effects on gut microbiota (Table 9.2). Of these additives,
organic acids and plant extracts are proposed as alternatives to antibiotic growth
promoters due to their antimicrobial properties.
TRIAL V
163
TRIAL II
might indicate that, contrary to thought, antibiotics could improve growth and gut
DISCUSSION
General discussion
Results obtained from trial IV showed a modulation of gut bacteria with sodium
the colonic bacterial ecosystem, although such as with antibiotics, when total
bacterial load was measured it did not change.
The change in similarity showed by the dendogram was clearly reflected in
LITERATURE
REVIEW
butyrate and plant extract; the dendogram obtained by RFLP confirmed changes in
changes in bacterial populations measured by qPCR with plant extract. It tended to
digesta. Increases in lactobacilli population by this plant extract have been shown
before (Manzanilla et al., 2004), although it is difficult to explain how this increase is
produced. Previous works have demonstrated a broad antibacterial activity for plant
extracts (Didry et al., 1994; Sen et al., 1998; Dorman and Deans, 2000); in particular,
OBJECTIVES
reduce enterobacteria population in jejunum and increased lactobacilli in caecum
for two of the extracts included in the mixture used: carvacrol from oregano (Dorman
Indeed, a potential supplantation of specific bacteria inhibited by the plant extract
(enterobacteria) by lactobacilli might be postulated. In this regard, recently, Si and
co-workers (2006) showed specific antibacterial activity of carvacrol and cinnamon
against E. coli. Also, modifications of gastrointestinal environment by a reduction of
fermentative activity in the small intestine by the extracts directly or by bacterial
shifts indirectly, could provide cecum and hindgut with a substrate with prebiotic
effect for lactobacilli. However, we could not detect changes in microbial activity in
the upper intestine measured as total bacteria, purine bases or microbial enzimatic
activities.
Contrary to plant extract, sodium butyrate effects observed on dendogram were
not reflected in lactobacilli or enterobacteria population, and presumibly this could
have been reflected in other bacterial groups (trial IV). Previous works of other
authors have found changes in ileal microbiota with decreases in coliform bacteria
parallel with increases in lactobacilli after administration of this sodium butyrate to
weaned pigs (Galfi and Bokori, 1990). Similarly, other works using formates have
also shown reductions in coliform bacteria (Øverland et al., 2000) and in total,
coliform and lactic acid bacteria (Canibe et al., 2005) throughout the gastrointestinal
tract.
However, these previous works detected high amounts of the organic acid
administered along the upper gastrointestinal tract that we could not confirm in our
study with increases only detected in the stomach (Manzanilla et al., 2006). Taking
164
TRIAL I
and Deans, 2000) and cinamaldehyde from cinnamon (Mancini-filho et al., 1998).
DISCUSSION
Chapter 9
this into account, and that RFLP results indicate changes in proximal colon
microbiota, some effect on stomach microbiota might somehow have modified the
LITERATURE
REVIEW
bacterial ecosystem in distal sections. In this regard, van Winsen and co-workers,
after administration of fermented liquid feed to growing pigs found decreases in
enterobacteria population in the stomach attributed to a higher population of
lactobacilli that could have limited their growth. Surprisingly, lower levels of
enterobacteria were maintained in feces where lactobacilli population were not
OBJECTIVES
different between diets. The authors attributed these results to some kind of carry over
effect of microbiota of anterior sections over posterior ones (van Winsen et al., 2001).
In addition, this lack of butyrate detection in the small intestine could be related to
the time of sampling, as Na-butyrate is readily absorbed in the gut starting in the
stomach (Bugat and Bentajac, 1993). In our case, animals were sacrificed between 46.5 h after limiting their access to feed, being therefore a potential factor affecting
TRIAL I
lack of acid in the small intestine (ad libitum access to feed from 20h to 8h). A
complete absorption of the acid at sacrifice time can therefore be behind the lack of
butyrate.
On the other hand, some kind of systemic effect of the butyrate absorbed can not
TRIAL II
9.3.3. Effects on microbiota by other mechanisms
Mode of action of some in-feed additives and feed strategies in gut microbiota
seems to be due to some kind of indirect effects on bacteria rather than direct
antibacterial activity. Indeed, modulation of gut environment, attachment sites and
TRIAL III
type/amount of substrate may be the key to the results found in some of the trials
included in the thesis.
In this regard, in trial V, a clear effect of mannan-oligosaccharides on
enterobacteria was observed. When piglets received the diet alone or in combination
with organic zinc, qPCR results showed a selective reduction in enterobacteria
TRIAL IV
counts, which was reflected in a higher lactobacilli:enterobacteria ratio. Similarly,
White and co-workers (2002) found a lower concentration of coliforms in the feces of
pigs fed diets with mannan-oligosaccharides. In this case, the effect as growth
promotant of this compound is related to a modulation of the gastrointestinal
ecosystem, reducing intestinal colonization by potentially pathogenic bacteria. This
TRIAL V
165
TRIAL II
be excluded (see chapter 9.4).
DISCUSSION
General discussion
modulation could be due to the fact that the oligosaccharide neutralizes binding
intestinal epithelium (Spring et al., 2000). However, a potential prebiotic effect of the
oligosaccharide, can not be discarded, and could also explain the effects observed.
However, in agreement with previous works (White et al., 2002), this hypothesis was
not supported by changes in the fermentation patterns measured as short chain fatty
LITERATURE
REVIEW
proteins on the surface of some bacteria and thus prevents their further attachment to
Apart from the different additives evaluated, in trial III, administration of
different types of fiber was also tested as a way to modulate the bacterial ecosystem.
It is well known that the modification of substrate reaching the lower gastrointestinal
tract is an effective tool to modify microbiota. However, controversies regarding fiber
OBJECTIVES
acids.
inclusion in the diet and apparition of some enteric diseases make this approach
In our case, the administration of diets rich in fiber (in form of resistant starch and
soluble and insoluble non-starch polysaccharides; trial III) did not result in marked
changes in bacterial groups studied along the gastrointestinal tract. Also, differences
in microbial activity measured as purine bases or enzymatic acitivities were not found
(data not shown). However, differences were shown with RFLP analysis, showing
that the animals fed with wheat bran had the lowest biodiversity in proximal colon
content and the most homogenous ecosystem between animals. In agreement,
Högberg and co-workers (2004) recently related administration of diets rich in
insoluble non-starch polysaccharides with a lower microbial biodiversity, which
might indicate a higher difficulty to digest this type of fiber resulting in a higher
specialization of bacteria inhabiting the lower gut. This implies therefore a lower
biodiversity that might be more easily interrupted by opportunistic pathogen
colonization.
These effects promoted by the different types of fiber could be explained not only
by increasing or changing the amount of substrate that arrives to be fermented but
also by differences reported for the same diets in some parameters such as digesta
viscosity, transit time and water binding capacity (Anguita et al., 2006). This might
also modify gut environment and thus impair or improve colonization by different
bacteria.
9.3.4. Other strategies to improve health and promote growth
166
TRIAL I
dubious (Hampson et al., 2001).
DISCUSSION
Chapter 9
Improvements in pig gut health and pig performance obtained when additives are
added to pig diets can not be completely explained by their effect on microbiota. An
LITERATURE
REVIEW
improvement of the piglet immune response, gut barrier and digestive capacity are
also considered as mechanisms by which some additives improve gut function and
performance. Among the additives tested, mannan-oligosacharides and organic zinc
(trial V) and sodium butyrate (trial IV) may be acting in these ways.
The administration of organic zinc (trial V) did not modify the bacterial groups
OBJECTIVES
studied, contrary to effects found when the mineral is added in inorganic form at
much higher concentrations (Katouli et al., 1999; Höjberg et al. 2005). However,
piglets showed a heavier empty ileum, considered as the section of the small intestine
showing a continuous Peyer’s Patch, which was also numerically longer. The higher
weight observed may be reflecting a higher development of the Peyer’s Patches. Also
IgA and IgM measured in jejunum digesta were numerically higher when organic
TRIAL I
zinc was administered, although differences did not reach significance.
Therefore the organic mineral could act by a different mechanism of action than
zinc oxide, with an immunoestimulatory effect, especially important at early’s stages
of piglet life when the immune system is still immature. In fact, it has been shown
TRIAL II
nonspecific immunity, and also for T and B lymphocytes proliferation (Shankar and
Prasad, 1998). Similarly, mannan-oligosaccharides have been reported to enhance the
pig immune response through activation of different membrane receptors by their
molecular similarity to different bacterial structures (Newman and Newman, 2001;
O’Quinn et al., 2001; Davis et al., 2004b). However, immune measurements done in
TRIAL III
our study did not show differences.
Moreover, a synergic effect was observed in intestinal morphology when organic
zinc and mannan-oligosaccharides were added together. Animals fed in both additives
together showed the lowest crypt depth and this was reflected in the highest
villus:crypt ratio which is considered an indicator of overall gut health (Zijlstra et al.,
1994). Similar results have been obtained in poultry with mannan-oligosaccharides
TRIAL IV
given alone (Iji et al., 2001; Ferket, 2002). This effect might be especially beneficial
at piglet weaning when villus atrophy and crypt hyperplasia appear with the
consequent impairment of gut function (Pluske et al., 1997). The reduction of
enterobacteria counts, and also a more suitable immune response might be behind
TRIAL V
167
TRIAL II
that Zn is crucial for the normal development and function of cells mediating
DISCUSSION
General discussion
these effects. Taking into account the relationship between the immune system and
In the case of sodium butyrate (trial IV), we have seen before that their
promoting effects on growth could be due to the observed effect on microbiota.
However, a systemic effect on pig health due to absorption of sodium butyrate
LITERATURE
REVIEW
gut bacteria, results obtained with both these additives seem particularly interesting.
administered in the diet can not be discarded, specially taking into account the fact
has a complex trophic effect on the gastrointestinal epithelium (Galfi and Bokori,
1990) by providing energy to epithelial cells (Bugat and Bentajac, 1993;
Cummings1995), improving absorption of sodium and water (Bond and Levit., 1976),
and also by stimulating proliferation index in crypts (Salminen et al., 1998). It is
OBJECTIVES
that acid was only detected in the stomach. It has been shown that sodium butyrate
possible therefore that butyrate was also acting in these ways, improving pig health
and thus explaining the improvement found in the gain:feed ratio and average daily
TRIAL I
gain (Manzanilla et al., 2006, In press).
9. 4. Summary
Results obtained in the different trials included in this thesis point to different
modes of action of additives tested. Antibiotic growth promoter, regarded as a model
to copy, did not act simply by reducing total bacterial load but probably by a more
complex modification of bacterial profile. In this regard, antibiotic strategy,
successfully and routinely used during the last fifty years, is not an easy strategy to
mimic.
Moreover, results obtained with the additives tested indicate different
mechanisms of action: selective effect on microbial groups, modulation of host health
by blocking adhesion of potential harmful bacteria, improvement of immune
response, or other systemic effects. Further studies are therefore required to improve
our knowledge regarding the exact mechanism of action of the different alternatives
to antibiotics proposed, although the achievement of an optimal equilibrium of gut
microbiota, and the improvement of gut function and immune response could be
considered the key responses to improve pig health and performance.
168
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L IV
TRIA
L III
TRIA
L II
DISCUSSION
Table 9.2. Summary of the main effects found for the different additives tested in the trials included in the thesis
Gut
Microbial indexesa
Trial
Additive tested
Intestinal
Total
section
bacteria
AB (avylamicin)
Biodiversity
Villus:
Crypt
Crypt
depth
Igs
═
═
═
═
XT (plant extract)
═
═
↓
AB (avylamicin)
═
═
═
≠
↑
═
═
═
≠
↑
═
↑
═
≠
↑
═
↓
═
═
═
═
═
═
═
↑
═
═
═
Jejunum
Caecum
XT (plant extract)
BM (mannan-oligosacharides)
BP’ (zinc-quelate)
BMP (mannan-oligosacharides
plus zinc quelate)
a
Profile
status
═
AC (sodium butyrate)
Trial V
morphology
═
AC (sodium butyrate)
Trial IV
Lactobacilli Enterobacteria
Immune
Jejunum
↓
A symbol has been assigned to classify effects observed on the different parameters evaluated (═, denotes abscence of changes; ↓, denotes a diminish
and ↑ denotes an increase compared to control diet).
═
TRIAL IV
TRIAL III
TRIAL II
TRIAL I
OBJECTIVES
170
CONCLUSIONS
Chapter 10
TRIAL V
INTRODUCTION
Conclusions
CONCLUSIONS
INTRODUCTION
CONCLUSIONS
OBJECTIVES
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
3. In the growing pig, the major bacterial groups quantified by fluorescent in situ
hybridization (FISH) differ along the gastrointestinal tract. Streptococci and
lactobacilli are the predominant in the upper tract, whereas
Bacteroides/Prevotella group, clostrial cluster XIV, IV and ruminoccoci are
the main groups in the lower tract.
4. The inclusion of coarse ground corn (4 mm), beet pulp (8%) or wheat bran
(10%) in the diet of growing pigs does not affect the main bacterial groups of
the intestinal tract. However, as observed by RFLP results, changes are
produced in the diversity of species within each group. In particular, wheat
bran, as a source of insoluble non-starch polysaccharides, promotes a decrease
in microbial diversity with more similar profiles between animals.
5. Contrary to what we expected, effects of avilamicyn on microbiota are related
to the modulation of its profile rather than a reduction in total bacterial load.
In particular, an increase in microbial diversity was demonstrated by RFLP
that could be behind the observed effects on performance.
INTRODUCTION
CONCLUSIONS
OBJECTIVES
TRIAL I
2. Commercial weaning produces a marked shift in piglet cecum microbiota,
with a significant decrease in the lactobacilli:enterobacteria ratio and changes
in bacterial profiles assessed by terminal restriction fragment length
polymorfism (t-RFLP). Specifically, a lower diversity in lactic acid bacteria
and the absence of some particular species like Lactobacillus delbruekii,
Clostridium butyricum and C. perfringens are related to microbiota disruption
by weaning.
TRIAL II
1. Real-time PCR used to quantify gut bacterial groups, in terms of 16S rRNA
gene copies, is a practical method to detect changes in microbiota equilibrium
by the lactobacilli:enterobacteria index. However, for absolute quantification
it generates higher counts than direct microscopy and selective culture.
TRIAL III
The results obtained in this thesis allow us to conclude that in our experimental
conditions:
TRIAL IV
Conclusions
172
TRIAL V
6. Similarly to avilamicyn, sodium butyrate and plant extract are able to modify
the microbial profile without modifying total microbial counts. However, each
INTRODUCTION
Chapter 10
CONCLUSIONS
additive promotes different changes. In particular, plant extract significantly
increases lactobacilli in the cecum, and butyrate promoted the highest
biodiversity.
OBJECTIVES
7. The growth performance improvement with mannan oligosaccharides and
organic zinc in weaning pigs are due to different modes of action. Whilst
mannan-oligosaccharides show an inhibitory effect on enterobacteria
population, organic zinc tends to improve development of the continuous
Peyer’s Patch. Synergy is manifested by a significant increase in villi:crypt
ratio when both additives are included together.
TRIAL I
TRIAL II
TRIAL III
TRIAL IV
TRIAL V
173
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TRIAL III
TRIAL II
TRIAL I
174
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Chapter 11
TRIAL V
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